CN116808238A

CN116808238A - Chiral virus-like nano-drug carrier capable of efficiently overcoming gastrointestinal physiological barrier and construction method thereof

Info

Publication number: CN116808238A
Application number: CN202310791542.4A
Authority: CN
Inventors: 李鹤然; 罗秋华; 杨华哲; 陈旭春; 王玉美; 桑振涛
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2023-09-29

Abstract

The invention discloses a chiral virus-like nano-drug carrier capable of efficiently overcoming gastrointestinal physiological barriers and a construction method thereof, wherein the chiral virus-like nano-drug carrier is a chiral virus-like mesoporous silicon drug carrier. The construction method of the chiral virus-like nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract comprises the following steps: indometacin is selected as a model drug, and indometacin IMC is loaded into series of mesoporous silicon spherical mesoporous silicon MSNs, viroid mesoporous silicon VSNs and chiral viroid mesoporous silicon CVSNs. The chiral virus-like mesoporous silicon has good degradability and biological safety, the virus-like topological structure and chiral modification can improve the surface interface activity of the carrier and show a certain nano-scale chiral recognition capability, the chiral virus-like mesoporous silicon shows remarkable advantages in each process of crossing intestinal barriers, can improve the functionality of the chiral virus-like mesoporous silicon serving as an oral drug carrier, has important research value and clinical transformation prospect, and provides reference for the surface interface design of future drug carriers.

Description

Chiral virus-like nano-drug carrier capable of efficiently overcoming gastrointestinal physiological barrier and construction method thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to a chiral virus-like nano-drug carrier capable of efficiently overcoming gastrointestinal physiological barriers and a construction method thereof.

Background

The oral administration is the current preferred mode of administration due to the advantages of good patient compliance, safety, non-invasive performance, convenient administration and the like. The nanoparticles are fully utilized in organisms, and the important point is to avoid being rapidly discharged from the body by the organisms. For oral formulations, when the drug or nanoparticle reaches the gastrointestinal tract, serious barriers including gastric acid environment, intestinal mucus, epithelial cell barrier and intestinal peristalsis down-stream gas flow, liquid flow and the like are faced, and the drug or nanoparticle needs to overcome the serious barrier to be absorbed into blood, so that the bioavailability is low and the absorption treatment effect is poor. Among these, the mucus and epithelial cell barriers are a major contributor to the inhibition of blood absorption by oral nanoparticles.

The intestinal mucosa epithelium is mixed with a large amount of goblet cells, and intestinal mucus is composed of water secreted by the goblet cells of the intestinal tract, mucin, carbohydrate, lipid and the like, and is a first defense line for the intestinal tract to resist pathogens and endogenous and exogenous stimuli. The mucus layer is a 3D reticular structure with viscoelasticity, so that the nanoparticles can be effectively captured and adsorbed, and then the nanoparticles are timely discharged along with rapid renewal of mucus, so that the nanoparticles have short residence time in vivo and cannot play a role. So that the mucus layer becomes an important obstacle for the absorption of the nano-particles in the gastrointestinal tract.

The tight junctions between intestinal epithelial cells, goblet cells, M cells and columnar epithelial cells constitute the intestinal epithelial cell barrier. The intestinal epithelial cells are not only responsible for absorption of nutrients into blood and metabolism, but also limit invasion and infection of foreign harmful substances, and are the main barriers affecting the absorption of oral medicines. Intestinal epithelial cells occupy the vast majority of the intestinal epithelial cell barrier, and the intestinal epithelial cell membrane is composed of lipid bilayer, which is a special structure that prevents many drugs from directly crossing the cell membrane, such as macromolecular substances that must enter the epithelial cells via receptor-mediated endocytosis.

Disclosure of Invention

In view of the above, the present invention discloses a chiral virus-like nano-drug carrier capable of effectively overcoming physiological barriers of gastrointestinal tract and a construction method thereof, so as to overcome the problems of low bioavailability caused by that the main barrier intestinal tract of oral absorption of drugs can greatly limit the oral transport efficiency of drugs and nanoparticles while maintaining the environmental balance in the intestinal tract.

The technical scheme provided by the invention is that in a first aspect, the invention provides a chiral virus-like nano-drug carrier for efficiently overcoming gastrointestinal physiological barriers, wherein the carrier is as follows: chiral virus-like mesoporous silicon drug carrier.

On the other hand, the invention also provides a construction method of the chiral virus-like nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract, which comprises the following steps: indometacin is selected as a model drug, and indometacin IMC is loaded into series of mesoporous silicon spherical mesoporous silicon MSNs, viroid mesoporous silicon VSNs and chiral viroid mesoporous silicon CVSNs.

Further, the method specifically comprises the following steps: the medicine carrying carrier is prepared by adopting a solvent volatilization method: firstly, preparing an IMC acetone solution with the concentration of 10mg/ml, weighing 60mg of MSN, VSN and CVSN samples, adding the samples into a brown bottle, and then adding 2ml of 10mg/ml indomethacin Xin Bingtong solution to enable the medicine/carrier to be 1/3w/w; sealing, stirring at 300rpm for 24h at room temperature after ultrasonic homogenization, volatilizing the solvent in a vacuum oven at 40 ℃ at an open mouth, and drying to obtain the IMC@MSN, IMC@VSN and IMC@CVSN medicine carrying carrier.

Further, the synthesis method of the MSNs comprises the following steps: sequentially adding 1g of CTAB and 1mL of ammonia water into a solution containing 100mL of deionized water and 30mL of ethanol, dropwise adding 3mL of TEOS after CTAB is completely dissolved, stirring for 4 hours at normal temperature, standing for 24 hours, centrifugally collecting a product, and washing with water and ethanol for multiple times; then, the sample was dried in vacuum at 45℃for 8h. And calcining in a muffle furnace at 550 ℃ for 6 hours to remove CTAB, thus obtaining the MSNs.

Further, the synthesis method of the viroid mesoporous silicon VSNs comprises the following steps: firstly, adding 40ml of deionized water, 1g of CTAB and 0.18g of TEA into a round-bottom flask, placing the flask in a water bath at 60 ℃ and stirring for 2 hours, and then dropwise adding 20ml of a mixed solution of cyclohexane TEOS, 16ml of cyclohexane and 4ml of TEOS; stirring in water bath at 60deg.C for 48 hr, transferring to oil bath at 98deg.C, stirring for 24 hr, centrifuging, collecting product, and washing with water and ethanol for several times; then, the sample was dried in vacuum at 45℃for 8 hours, and then calcined in a muffle furnace at 550℃for 6 hours to remove CTAB, thereby obtaining VSNs.

Further, the synthesis method of the chiral viroid mesoporous silicon CVSNs comprises the following steps:

synthesis of AVSNs: performing surface amination modification on the VSNs by using APTES, weighing 100mg of VSNs with template removed, performing ultrasonic dispersion in absolute ethyl alcohol, adding 300 μl of APTES, stirring in a water bath at 50 ℃ for 24 hours, washing with absolute ethyl alcohol for multiple times, centrifuging, drying, and collecting samples to obtain amino-functionalized VSNs, namely AVSNs;

the surface chiral modification was accomplished by grafting chiral molecule L/D-alanine by acylation reaction, and 20mg of AVSNs was dispersed in 5ml of anhydrous DMSO. Adding 81.27mg of Boc-L/D-alanine into the mixed system in the presence of EDCI/HOBT, stirring at room temperature for 48 hours, centrifuging, washing alternately with water/alcohol, and collecting a sample after drying to obtain Boc-L/D-AVSNs;

In order to remove the Boc protecting group, trifluoroacetic acid (TFA) is used for treatment, 15mg of Boc-L/D-AVSNs are weighed and uniformly suspended in 15ml of DMSO, 6.53ml of TFA is added for reaction at room temperature for 3 hours, centrifugation, absolute ethyl alcohol washing and drying are carried out, and the L/DVSNs are obtained, wherein LVSN is CVSN.

The invention provides a chiral virus-like nano-drug carrier capable of efficiently overcoming gastrointestinal physiological barriers and a construction method thereof. The L-alanine is modified by an amide bond, so that the chirality of the virus-like mesoporous silicon is endowed, the adhesion to organisms and the specificity recognition potential of the chirality are increased, and a virus-like chiral mesoporous silicon nano drug delivery system capable of efficiently breaking through an intestinal barrier is successfully constructed.

The chiral virus-like mesoporous silicon has good degradability and biological safety, the virus-like topological structure and chiral modification thereof can promote the surface interface activity of the carrier and show a certain chiral recognition capability of nanometer scale, the chiral virus-like mesoporous silicon shows remarkable advantages in each process of crossing intestinal barriers, can promote the functionality of the chiral virus-like mesoporous silicon serving as an oral drug carrier, has important research value and clinical transformation prospect, and provides references for the surface interface design of future drug carriers.

VSN and CVSN are used as carriers, and the distribution of the drugs in the body can be changed by depending on the functional and chiral characteristics of topological structure advantages, and particularly, the time of the drugs in the body can be prolonged, so that the drug has higher bioavailability than the traditional spherical mesoporous silicon, and further has good anti-inflammatory pharmacodynamics. The method provides a new idea for solving the common problems of the medicines with low dissolution rate, less absorption, low bioavailability and the like of the insoluble medicines.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure of the invention as claimed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a series of graphs of TEM, SEM and particle size analyses of silicon nanoparticles according to an embodiment of the disclosure;

FIG. 2 is a graph of the series of silicon nanoparticles provided by the disclosed embodiments of the invention, including (A) an infrared spectrogram, (B) a circular dichroism spectrum, and (C) a thermogravimetric analysis;

FIG. 3 shows (A) a nitrogen adsorption curve, (B) a BJH pore size distribution curve, and (C) a SAXS spectrum of a series of silicon nanoparticles provided by the disclosed embodiments of the present invention;

FIG. 4 shows the Zeta potential of the initial contact angle (B) of the interface characteristic (A) of the series mesoporous silicon surface according to the embodiment of the present invention;

FIG. 5 is a graph showing amino acid adsorption characteristics (A) and amino acid adsorption CD spectrum (B) according to the disclosed embodiment of the invention;

FIG. 6 is a graph showing degradation rates of a series of carriers in different simulated fluids (A) simulated gastric fluid (B) simulated intestinal fluid (C) simulated body fluid provided in accordance with an embodiment of the present disclosure;

FIG. 7 shows the hemolysis ratio of hemolysis photograph (B) of the carrier hemolysis test result (A) provided by the disclosed embodiment of the invention;

FIG. 8 shows the adsorption of BSA protein by a series of carriers provided in the disclosed embodiments of the present invention;

FIG. 9 is a series of vector cytotoxicity provided by an embodiment of the present disclosure;

FIG. 10 is a graph showing the rate of body weight gain after oral administration of a series of vehicles provided in accordance with an embodiment of the present disclosure;

FIG. 11 is an in vivo toxicity blood biochemical test of a series of vectors provided in accordance with an embodiment of the present disclosure;

FIG. 12 is a graphical representation of in vivo toxicity blood normative index for a series of vectors provided in accordance with an embodiment of the present disclosure;

FIG. 13 is a major organ tissue section of control group and oral administration series vehicle mice provided in the disclosed embodiment of the invention;

FIG. 14 shows the intestinal adhesion (A) in vivo imaging and the ROI intensity (B) intestinal adhesion rate of a series of carriers provided in the disclosed embodiments of the present invention;

FIG. 15 is a three-dimensional scan image of mucus penetration provided by an embodiment of the present disclosure;

FIG. 16 is a series of carrier gastrointestinal retention capacities (A) intestinal retention in vivo imaging (B) stomach relative fluorescence intensity (C) small intestine site relative fluorescence intensity provided by an embodiment of the present disclosure;

FIG. 17 is a graph showing in vivo imaging distribution and relative fluorescence intensity of a series of vectors provided in the disclosed embodiments in mice heart, liver, spleen, lung, kidney and brain;

FIG. 18 shows nanoparticle absorption by intestinal tissue after 2h of oral administration provided by the disclosed embodiments of the present invention;

FIG. 19 is an ultraviolet absorption spectrum of an IMC in different media provided in accordance with an embodiment of the present disclosure;

FIG. 20 shows the pore size distribution of the nitrogen adsorption and desorption curve (B) of the drug carrier (A) provided by the disclosed embodiment of the invention;

FIG. 21 is an infrared spectrum of a carrier before and after drug loading according to an embodiment of the present disclosure;

FIG. 22 is an XRD pattern of an IMC, blank carrier, drug carrier and IMC carrier mixture provided in accordance with an embodiment of the present disclosure;

FIG. 23 is a DSC of an IMC, blank carrier, drug carrier and IMC carrier mixture provided in an embodiment of the present disclosure;

fig. 24 is a graph showing the initial contact angle (a) and the contact angle (B) of a drug carrier according to an embodiment of the present disclosure;

FIG. 25 is a graph showing the release profile of IMC in various dissolution media (A) pH 6.5 (B) pH 6.8 (C) pH 7.4 provided by the disclosed embodiments of the present invention;

FIG. 26 is a graph of time versus drug concentration for IMC, IMC@MSN, IMC@VSN and IMC@CVSN provided by an embodiment of the present disclosure;

FIG. 27 shows in vivo distribution of IMC, IMC@MSN, IMC@VSN and IMC@CVSN at different times (A) 1h (B) 3h (C) 6h according to an embodiment of the present disclosure;

FIG. 28 is a plot of swelling ratio versus time for treating rat ankle swelling for saline, IMC drug substance, IMC@MSN, IMC@VSN, IMC@CVSN provided in an embodiment of the present disclosure;

fig. 29 is a view showing the appearance of a rat ankle swelling area and a pathological section of skin tissue according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the invention. Rather, they are merely examples of systems consistent with aspects of the invention as detailed in the accompanying claims.

The intestinal barrier is the main barrier for oral absorption of the drug, can greatly limit the oral transport efficiency of the drug and the nanoparticles while maintaining the environmental balance in the intestinal tract, and is the main reason application for causing the low bioavailability. The high-efficiency invasion capability of the virus has certain advantages in the processes of infiltration, adhesion, invasion and the like, and the embodiment utilizes the morphological advantages of the virus and the physicochemical characteristics of the mesoporous silicon to construct a drug delivery system, and efficiently spans a physiological barrier by depending on the structural advantages of the drug delivery system. Meanwhile, because endogenous substances such as DNA, amino acid and the like forming a living body often have chiral characteristics, the biological behavior of mesoporous silicon in the body is improved by modifying chiral micromolecular L-alanine on the basis, the adhesion and chiral specific recognition potential of a biological interface are increased, and a virus-like chiral mesoporous silicon imaging drug delivery nano system which aims at breaking through an intestinal barrier is constructed.

Example 1

To verify the advantages of the viroid mesoporous silicon morphology in the aspect of being used as a drug carrier, the embodiment synthesizes smooth Solid Silicon Nanoparticles (SSNs), spherical Mesoporous Silicon (MSNs), viroid mesoporous silicon (VSNs) and chiral viroid mesoporous silicon (CVSNs) at the same time, and the synthesis method is as follows:

synthesis of SSNs: in a solution containing 40mL of ethanol, 3mL of deionized water and 0.8mL of ammonia water, 2mL of LTEOS was added dropwise, stirred at normal temperature for 12 hours, and the product was collected by centrifugation and washed with water and ethanol several times. Then, the sample was dried in vacuum at 45℃for 8h.

Synthesis of MSNs: in a solution containing 100mL deionized water and 30mL ethanol, sequentially adding 1g CTAB and 1mL ammonia water, dropwise adding 3mL TEOS after CTAB is completely dissolved, stirring at normal temperature for 4 hours, standing for 24 hours, centrifugally collecting a product, and washing with water and ethanol for multiple times. Then, the sample was dried in vacuum at 45℃for 8h. And then calcining in a muffle furnace at 550 ℃ for 6 hours to remove CTAB, thus obtaining the final sample.

Synthesis of VSNs: first, 40ml of deionized water, 1g CTAB and 0.18g TEA were added to a round bottom flask. After the flask was placed in a 60℃water bath and stirred for 2 hours, 20ml of a mixed solution of cyclohexane TEOSd (16 ml of cyclohexane, 4ml of TEOS) was added dropwise, and after stirring in a 60℃water bath for 48 hours, the mixture was transferred to a 98℃oil bath and stirred for 24 hours, and the product was collected by centrifugation and washed with water and ethanol several times. Then, the sample was dried in vacuum at 45℃for 8h. And then calcining in a muffle furnace at 550 ℃ for 6 hours to remove CTAB, thus obtaining the final sample.

Synthesis of AVSNs: and (3) carrying out surface amination modification on the prepared VSNs by using APTES. 100mg of VSNs (template removed) were weighed and dispersed ultrasonically in absolute ethanol. After 300. Mu.l APTES was added, the mixture was stirred in a 50℃water bath for 24 hours, washed with absolute ethanol several times, centrifuged, dried, and the sample was collected to obtain amino-functionalized VSNs, namely AVSNs.

Synthesis of L/DVSNs: the chiral molecule L/D-alanine is grafted through an acylation reaction to finish the surface chiral modification. 20mg of AVSNs were dispersed in 5ml of anhydrous DMSO. In the presence of EDCI/HOBT, 81.27mg of Boc-L/D-alanine is added into the mixed system, stirred for 48 hours at room temperature, centrifuged, washed alternately with water/alcohol, and dried to collect samples, thus obtaining Boc-L/D-AVSNs.

And (3) taking a proper amount of SSNs, MSNs, VSNs and CVSNs powder samples, uniformly dispersing the powder samples in absolute ethyl alcohol by ultrasonic, coating the powder samples on a micro-grid copper sheet coated with a carbon film, and performing projection electron microscope (TEM) analysis after drying in an oven. The support powder was metal sprayed for Scanning Electron Microscope (SEM) analysis. And (3) taking a proper amount of SSNs, MSNs, VSNs and CVSNs powder, performing ultrasonic dispersion in absolute ethyl alcohol, and performing particle size distribution and particle size analysis by using DLS.

The results of TEM, SEM, and particle size analysis of the above-described series of silicon nanoparticles are shown in FIG. 1.

The SSNs are of a monodisperse smooth nonporous nanosphere structure, the MSNs are of a monodisperse rough mesoporous sphere structure, the VSNs are of a monodisperse virus-like mesoporous carrier, the surface of each VSNs is provided with a large number of uniformly distributed vertical nanotubes, and meanwhile, the CVSNs also have virus-like morphology characteristics after grafting chiral molecules L-alanine. The four are uniform in appearance and basically the same in size, the particle size imaging statistics result is between 90 and 100nm, and the hydration particle size measured by DLS is slightly larger than the true value and is between 130 and 140nm due to the influence of hydration.

The infrared spectrogram, circular dichroism spectrum and thermogravimetric analysis curve of the series of silicon nanoparticles are shown in figure 2.

CVSNs infrared spectrogram is 1681cm ^-1 The carbonyl stretching vibration peak of the amide group is reserved, which indicates that the chiral L-alanine molecular grafting is successful. In addition, CVSNs have infrared spectrum of 462cm ^-1 The Si-O-Si bending vibration peak was shown at 1087cm ^-1 The Si-O-Si asymmetric stretching vibration peak is shown. The L-alanine grafted VSNs and the D-alanine grafted VSNs showed positive and negative cotton effects in the range of 210-250 nm, and no obvious chiral response signal was observed for the VSNs, suggesting that alanine chiral modification was successful. SSNs, MSNs, VSNs and CVSNs have weight loss of 1.20%,4.98%,9.76% and 24.07% respectively in the range of 25-700 ℃, and the grafting amount of chiral micromolecule L-alanine can be estimated to be about 14.31%, which indicates that the CVSNs specifically graft chiral micromolecule successfully.

The SAXS patterns of the series of silicon nanoparticles before and after the removal of the templates are shown in FIG. 3, along with the nitrogen adsorption curve, BJH pore size distribution curve, and MSNs and VSNs.

MSNs, VSNs, CVSNs all showed distinct adsorption/desorption isotherms of type IV and contained hysteresis loops indicating the presence of a uniform mesoporous structure. The pore diameter test results show that all three have mesoporous pores, and specific numerical values are shown in table 1. The pore sizes are respectively as follows: 3.2nm, 2.9nm, 2.5nm. Solid, non-porous SSNs do not observe type IV adsorption and desorption isotherms and have no mesoporous grade pore size distribution. SAXS results in the 0.1-5 (2-theta) test range indicate that VSNs and MSNs have higher diffraction peaks after template removal, i.e., higher order of pore structure, than before template removal.

TABLE 1 specific surface area, pore volume and pore size of silicon nanoparticles

Surface interface properties of the series of silicon nanoparticles: the Zeta potentials of SSNs, MSNs, VSNs, AVSNs and CVSNs are 2.67+/-1.11, -2.36+/-0.19, -13.67+/-4.06, 35.15 +/-3.6 and 21.16+/-1.36 mV respectively, and the SSNs and MSNs have similar surface characteristics and exhibit electric neutrality; VSN has a weak negative charge; the potential of the AVSNs modified by the amino is greatly increased, and the AVSNs modified by the amino is positive, which indicates that the amino modification is successful; CVSNs have increased surface potentials compared with VSNs, demonstrating successful grafting of chiral molecules. The initial contact angles of SSNs, MSNs, VSNs and CVSNs are 43.53 degrees, 30.56 degrees, 19.70 degrees and 14.45 degrees respectively, and the contact angles of VSNs and CVSNs are reduced to 0 degrees in a very short time, so that the surface roughness of a carrier surface can be obviously increased through both virus topological structure and chiral grafting.

Amino acid adsorption properties of chiral viral silicon: the results of the amino acid adsorption characteristics are shown in FIG. 5, and the CD spectrum chart shows the corresponding symmetric cotton effect in the L/D alanine adsorption experiment of the chiral modified virus-like mesoporous silicon with different configurations. It can be concluded that LVSNs have a greater adsorption capacity for L-alanine than D-alanine; similarly, DVSNs have a greater adsorption capacity for D-alanine than L-alanine.

The rough spinous process interface of the VSNs in this embodiment has more contact sites, larger outer surface area and high interfacial reactivity than the spherical carrier. Compared with SSNs and MSNs, VSNs are assembled by monodisperse micelles by virtue of outer clusters, the pore diameter is smaller, the capillary action in the pores is stronger according to a kelvin equation, larger additional pressure can be generated on liquid, and the water contact angle is obviously reduced. And secondly, the VSNs interface has high affinity, a multi-site rough topological structure, the wetting process is easier to occur, the mass transfer of liquid to the inside of the carrier is promoted, the adsorption capacity of the carrier interface is also obviously enhanced, the cell uptake or signal transduction can be further promoted, the biological behaviors such as contact, adhesion and the like of the VSNs are enhanced, and the in vivo application of the VSNs as a drug carrier is facilitated. In addition, the amino acid chiral modification enhances the hydrophilicity of the surface of the carrier, the contact angle of CVSNs is further reduced relative to VSNs, the CVSNs has selective adsorption property, the adsorption capacity to L-type amino acid is obviously stronger than that of D-type amino acid, and the chiral recognition capacity of a certain nano-scale surface interface is shown.

Example 2

Degradation of the above-mentioned series of vectors

The degradation trend of the series of vectors in SGF, SIF and SBF is shown in figure 6.

From the figure, the series of carriers are slowly degraded in all three simulated liquids, the degradation rate is faster in the first week, and the degradation rate is slower in the following 2-12 weeks than in the first week. The different pH values of the degradation simulation liquid can obviously influence the explanation process, and the degradation speed is as follows: SBF > SIF > SGF. In addition, VSN has a faster degradation rate relative to SSN, MSN; the degradation rate of the chiral modified CVSN can be further improved on the basis of VSN.

Example 3

Hemolysis test of series of vectors

The hemolytic image and the hemolysis ratio are shown in FIG. 7.

The results show that the hemolysis rate of the series of silicon nano-carriers has concentration dependency, the hemolysis rate rises with the increase of the concentration, and when the concentration reaches 100 mug/ml, the MSN hemolysis rate exceeds 10%. In contrast, the hemolysis rates of SSN, VSN and CVSN only increase slightly along with the increase of the concentration, are smaller than 7% in the concentration range of 5-100 mug/ml, have low hemolysis rate and good blood compatibility, and all meet the requirements of national standards on the hemolysis of biological materials. In addition, the hemolysis rate is reduced slightly after chiral modification of L-alanine.

Example 4

BSA protein of series carrier the BSA protein adsorption results are shown in FIG. 8. The weaker the adsorption of the material to the protein, the less it affects the balance of anticoagulant/procoagulant factors in the broken blood, the higher the safety. The adsorption of BSA by each of the above carriers showed that the adsorption of BSA by each of SSNs (1.6%), MSNs (8.1%), VSNs (6.7%) and CVSNs (9.5%) was less than 10%, which means that the carriers were insensitive to proteins in blood and had good biosafety. Compared to MSNs (8.1%), VSNs have significantly reduced adsorption to BSA because of the electrostatic repulsion of the negative potential surface of VSNs with the negative charge carried by the protein; the CVSNs have higher adsorption capacity than VSNs proteins, and on the one hand, the surface potential can be increased, and on the other hand, the affinity to BSA can be increased after the L-alanine is surface-modified, so that the CVSNs have higher adsorption capacity.

Example 5

Cytotoxicity of the series of vectors is shown in figure 9; the results show that SSNs, MSNs, VSNs and CVSNs vectors have no significant effect on the survival rate of Caco-2 cells in the concentration range of 0.1-100 mug/ml after 24, 48 and 72 hours of culture of the vectors and the cells respectively. Even if the concentration of the carrier is far greater than the usual dose of 100 μg/ml for clinical use, the cell viability is still higher than 80%. Within the range of the study dose, the cell viability was not significantly dependent on the vector dose. The results prove that the vector has good biological safety, does not generate obvious cytotoxicity on Caco-2 cells, and has no obvious difference on the influence on the cell survival rate.

Example 6

In vivo toxicity experiments for the series of vehicle-body weight gain, mice body weight gain curves after oral administration are shown in figure 10. The experimental results showed that all mice did not show any abnormal clinical behavior throughout the experimental interval (including the adaptation period of the previous week), with the placebo group mice having a weight gain of 36.4%, the SSN, MSN, VSN and CVSN group mice having weight gains of 39.1%, 32.1%, 39.3% and 38.2%, respectively. The results show that the weight gain of mice is not affected by the carrier materials of each group, and the weight gain of mice in the experimental group is not obviously different from that of mice in the blank control group.

Example 7

The in vivo toxicity test-blood biochemical index and blood routine result of the series of vectors are shown in fig. 11 and fig. 12, respectively. After continuous administration for 15d, each blood routine index is in the reference range of ICR mice, and each index between the mice in the experimental group and the mice in the control group has no obvious difference, which indicates that SSNs, MSNs, VSNs and CVSNs carriers can not cause abnormality of blood components in the test interval. Figures 2-9 show that blood biochemical parameters after administration are also within the reference range of ICR mice, and that there is no obvious difference in each index between the experimental group and the control group mice, indicating that the vector will not cause damage to liver function and kidney function in the test interval. The in vivo safety experiment result shows that the SSNs, MSNs, VSNs, CVSNs carrier has good biocompatibility and low toxicity, and meets the basic requirements of clinical application of biological materials.

Example 8

In vivo toxicity experiments of the series of vectors, namely, the dirty body ratio and tissue sections, the dirty body ratio results are shown in the in vivo toxicity dirty body ratio of the series of vectors in Table 2, and the organ tissue section results are shown in FIG. 13.

TABLE 2 series in vivo toxicity to dirty body ratio of vectors

After dissecting the organs, the phenomena of organ enlargement, abnormal color and the like are not found. The organ/body weight ratios of the series of vehicle sample groups and the blank group mice were not significantly different, and were all within the normal range of ICR mice. As a result of the tissue section, no local inflammation, injury, necrosis and the like were observed. The SSNs, MSNs, VSNs, CVSNs instructions all have good biosafety.

The above examples relate to degradability, cytotoxicity and in vivo and in vitro toxicity, respectively, of the series of vectors. The carrier is influenced by the action of water molecules and conditions such as ions, enzymes, temperature and the like in a physiological environment, so that the phenomena of integrity loss, property change or performance reduction are caused; meanwhile, because the abundant silicon hydroxyl groups on the surface of the nano silicon form hydrogen bonds with water molecules in the degradation liquid, the skeleton fracture and the material dissolution are promoted, the weight loss is gradually increased along with the degradation time, and the carrier material is degraded. The degradation experiment result shows that: degradation rate SSNs < MSNs < VSNs, probably because of the effect of the morphological features of the VSNs, the rough spinous processes on the surface of the VSNs give it a large number of contact sites, active interfacial properties, and according to the better wettability of the VSNs described above, a smaller contact angle with the degrading medium, a larger contact area with the liquid, a faster degradation rate, but still maintain the functionality of the support in various simulated media. In addition, VSNs have smaller pore sizes, kelvin acts more strongly, and can create greater additional pressure on the wetting fluid, promoting mass transfer of the degrading fluid into the interior of the support. Besides the structure of the material, the degradation process is also influenced by the pH of a degradation medium, and the degradation speed of the SSNs, the MSNs and the VSNs is increased along with the increase of the pH, so that the degradation speed is slower in gastric juice, the rigid structure of the carrier is not damaged, and the drug delivery to the small intestine part is protected to be absorbed orally.

In conclusion, the virus silicon is slowly degraded in body fluid, so that effective physical protection is provided for the loaded medicine in the process of transferring and releasing, and the carrier can be completely degraded after the completion of the service, so that the potential safety hazard caused by long-time retention of the carrier in vivo is avoided. No matter what way (oral administration, injection) the nanocarrier is administered, it is inevitable that the nanocarrier will come into contact with blood, so blood safety is an important index for evaluating the nanomaterial. In the hemolysis experiment, although VSNs have rough spinous process surfaces, they show lower hemolysis (< 5%) at clinical concentrations (5-100 μg/mL) and the hydrophilicity of the carrier is enhanced by modification with L-alanine to shield Reactive Oxygen Species (ROS) on the surface, thus playing a role in reducing hemolysis. The hydrophilic silicon hydroxyl groups on the surface of the mesoporous silica carrier can be combined with albumin through hydrogen bonding, so that the anticoagulation effect of the albumin is affected, and potential coagulation or thrombus formation tendency is realized. In the protein adsorption experiment, the adsorption amount of SSNs, MSNs, VSNs and CVSNs proteins is below 10%, wherein CVSNs is increased compared with VSNs proteins in adsorption amount, and the reason is probably that after the L-alanine is surface modified, the affinity of the CVSNs with proteins is improved. In cytotoxicity experiments, the cell survival rate, the culture time and the carrier concentration of the serial carriers and cells after co-culture have no obvious dependence, and no cytotoxicity can be generated. In vivo toxicity studies showed that the animals did not develop any abnormal clinical manifestations throughout the duration of the experiment. There was no significant difference in weight gain between the different vehicles. After organs (heart, liver, spleen, lung and kidney) were collected, no obvious swelling or inflammation was observed, and the visceral ratios were all in the normal range. Tissue section examination showed that each organ was characterized by distinct structural features, distinct tissue morphology, no structural changes and no local inflammation and necrosis were seen, and no distinct differences were seen between the groups. There is no obvious difference between blood routine test index and liver and kidney function biochemical test result, and the measured values are all in normal reference range.

In vitro toxicity of the series of vectors was assessed by blood safety, cytotoxicity, in vivo toxicity of the vectors was assessed by recording clinical manifestations, body weight and body weight ratios, blood norms, blood biochemical indices and vital organ tissue sections. The results show that the VSNs and CVSNs used as drug carriers have good safety in vivo and in vitro and can be safely applied to the construction of drug delivery systems.

Example 8

Ex vivo intestinal bioadhesion the results of the ex vivo intestinal bioadhesion of the series of vectors are shown in figure 14. The premise of oral absorption of the drug carrier is sufficient contact with the intestinal tract. Theoretically, the large contact area of the carrier and the small intestinal mucosa is beneficial to oral absorption of the medicine. The picture results show that the carrier adhesion rate of the SSN is only about 40% after 5min of elution, the MSN adhesion rate is slightly improved relative to the SSN, and the adhesion rate is 55% after the elution process is finished. And in the three types of carriers with different shapes and structures, the retention capacity of VSNs on intestinal mucosa is obviously enhanced compared with MSNs and SSNs, and more than 80% of the carriers can be adhered to intestinal tissues, so that good bioadhesion is shown. As can be seen from panel B, the bioadhesion capacity of the carrier is closely related to morphology, and the order of the adhesive capacities of the three carriers is VSNs > MSNs > SSNs. In addition, CVSNs still have a small enhancement in adhesion to VSNs due to better wettability and smaller pore size, while surmising that their surface-modified L-alanine produces chiral response recognition.

Example 9

Intestinal mucus permeability the results of intestinal mucus permeability of the series of vehicles are shown in figure 15. Wherein green fluorescence is a FITC-labeled mucus layer, and red fluorescence is RITC-labeled silicon nanoparticles.

Example 10

Intestinal retention the results of intestinal retention of the series of vectors over time are shown in figure 16. For SSNs, a weaker fluorescent signal was observed in the stomach and a stronger fluorescent signal was observed at the small intestine after 1h of oral administration; the fluorescence intensity at the small intestine is obviously reduced at 2 hours compared with that at 1 hour, and almost completely disappears at 6 hours, and the results show that the smooth and nonporous SSNs have small contact area, small friction force, extremely high discharge speed and short retention time in the intestinal tract. Whereas MSNs detected fluorescent signals in both stomach and small intestine tissues 1h after dosing; after 2 hours, fluorescence signals were observed only in the intestinal tract, and the fluorescence signals were mainly concentrated in the middle-rear segment of the small intestine and the large intestine, fluorescence signals were observed only in the large intestine, i.e., intestinal excretion, but not in the small intestine absorption sites, and at 12 hours, metabolism was substantially complete in the intestinal tract, and no strong fluorescence was observed. In contrast, the fluorescence intensity of the VSNs at the corresponding time points in the small intestine is obviously stronger than that of the SSNs and the MSNs, and a stronger fluorescence signal can be captured after 12 hours of administration, so that the VSNs have longer retention time in the intestinal tract. For CVSNs, the fluorescent signal is mainly concentrated in the stomach at 1h after administration, the intestinal tract has only weaker fluorescent signal, and CVSNs show stronger fluorescent signal intensity than VSNs at various time points over time. The above results correspond to bioadhesion experimental results of the isolated intestinal segment.

Example 11

In vivo distribution of the series of silicon nanoparticles the results of in vivo distribution of the series of silicon nanoparticles are shown in figure 17. The results show that the fluorescence signals of the VSNs and CVSNs vectors are higher than those of SSNs and MSNs in almost every organ, and that the distribution intensity of the two vectors in heart, lung, brain over time is significantly higher than that of liver, spleen, kidney for VSNs and CVSNs. Fluorescence intensity in the lung and brain in combination with imaging and ROI fluorescence intensity analysis: CVSN > VSN > MSN > SSN, it is speculated that VSNs and CVSNs have the potential to target the brain and lungs. For CVSNs, the fluorescence intensity of each organ reaches a maximum value when orally administered for 1h, and the fluorescence intensity drops rapidly in 1-2 h. In summary, VSNs and CVSNs have significant advantages over SSNs and MSNs in vivo distribution.

Example 12

The intestinal villi absorption of the series of vectors is shown in figure 18. The ability of the nanoparticles to overcome both mucus and intestinal epithelial cell barriers can be visualized by observing the distribution of fluorescent-labeled nanoparticles in ICR mouse intestinal sections: CVSNs > VSNs > MSNs > SSNs.

The mucus layer and the small intestinal epithelial cell layer form an intestinal mucosa layer of the digestive tract, and as can be seen from the image, the red fluorescence signal of the SSNs is extremely weak, and is mainly concentrated in the center of the intestinal cavity, but almost no red fluorescence can be observed near the small intestinal villi, which indicates that the SSNs are difficult to stay in the intestinal tract to permeate the intestinal mucosa barrier; the fluorescence intensity of MSNs at the small intestinal mucosa is slightly enhanced and distributed more widely than that of SSNs, but red fluorescence-marked nano particles rarely enter the small intestinal villus and mainly exist in the small intestinal villus gap; compared with SSNs and MSNs, VSNs have high fluorescence intensity at small intestinal mucosa, are mainly distributed around small intestinal villi and can enter the small intestinal villi; in addition, the red fluorescence of the L-alanine modified CVSNs is uniformly distributed around the villus of the small intestine and the fluorescence signal is obviously enhanced, and meanwhile, the very strong fluorescence signal exists in the villus of the small intestine, so that the results show that VSNs and CVSNs can penetrate through the intestinal barrier and absorb blood through the villus of the small intestine.

The nano carrier is important to make full use of in vivo things, and is important to avoid being rapidly cleared by organisms, so that the prolonging of the residence time of the medicine carrier at the absorption part is important. In intestinal adhesion experiments, VSNs and CVSNs show good bioadhesion, and the rough spinous process surface of viral silicon has high reactivity and can provide a large number of contact sites for anchoring biological tissues when in contact with tissue interfaces.

The mucus layer of the gut is a difficult physiological and mechanical barrier to cross for oral nanoscale drug carriers. In addition, the cup cells in the small intestine can continuously secrete new mucus, and meanwhile, the peristaltic motion of the intestinal tract can promote the timely renewal and discharge of the mucus, and if the medicine carrier cannot quickly permeate into the epithelial cells at the bottom of the mucus layer, the medicine carrier faces the destiny. In mucus permeation experiments, SSNs are mainly concentrated on the upper layer of mucus near the intestinal lumen side, almost no particles cross the mucus barrier, and the mucus layer is seen to be an important barrier limiting the absorption of nanoparticle oral routes. The MSNs are slightly more distributed in the mucus layer than SSNs, and some particles penetrate the mucus to the epithelial cell layer, but still fail to achieve effective longitudinal penetration in the z-axis direction. Whereas VSNs with a virus-like morphology have a rough spinous process surface, are in multi-site contact with the mucus layer, diffuse deep in the mucus layer and are widely distributed, exhibit better mucus penetration than SSNs and MSNs, and effectively overcome this barrier. The rough surface spinous process is used as an interface for breaking through organisms, a large number of curved recognition sites are generated at the joint of the rough surface spinous process and the biological membrane, and the geometric centers can attract corresponding proteins, lipids and the like to gather and further evolve into bioactive centers to mediate cell recognition or signal transduction, so that the rough surface spinous process has positive promotion effects on biological behaviors such as mucosa adhesion, cell uptake and the like. And the method also provides greater possibility for improving the bioavailability of the loaded drugs subsequently, and in addition, VSNs and CVSNs have stronger bioadhesion capability and longer in-vivo retention time. The distribution of different carriers in the body after absorption into blood through intestinal tracts also shows a certain difference, which suggests that the affinity of the carriers with different organs is different possibly due to the morphology, chiral modification and other reasons of the carriers. The VSNs and CVSNs can remarkably improve the distribution of the vector in the lung, brain and kidney of animals, have potential application value in the aspect of delivering medicaments for treating diseases such as the lung, the brain and the kidney, and can also provide thought for organ targeted release of the medicaments.

In small intestinal villus absorption experiments, SSNs are mainly concentrated on the intestinal lumen side, and almost no red fluorescence distribution near villi is caused by weak mucous permeability, which indicates that small intestinal villus absorbs very poorly on smooth solid silicon spheres. MSNs have a coarser porous surface than SSNs and are widely distributed in the intestinal lumen, but are mostly distributed in the small intestinal villus space, and no fluorescence distribution is seen in the small intestinal villus. VSNs have high fluorescence intensity at the small intestinal mucosa compared to SSNs and MSNs, are mainly distributed around and can enter the small intestinal villi. The above results demonstrate that nanoparticle surface roughness is an important factor affecting nanoparticle penetration across the intestinal mucosal barrier, i.e., the viral-like multi-contact nanoparticles have better intestinal mucosal penetration. In addition, the L-alanine modified CVSNs can be provided with obvious fluorescence distribution on the villus side and in the villus interior of the small intestine, and the CVSNs have unique virus-like rough spinous process surface and chiral modification, so that the absorption capacity of the small intestine villus is improved.

Example 13

The drug loading rate and the drug loading rate of the drug carrier are shown in table 3.

TABLE 3 drug loading and drug loading rates for different drug carriers

Example 14

Status and Properties of drug in Carrier

After loading IMCs, the specific surface area, pore volume and pore diameter of MSNs, VSNs and CVSNs were changed, and thus the drug carrier was tested for specific surface area, pore volume and pore diameter, and the results are shown in fig. 20 and table 4.

From the results, after the IMC is loaded into the carrier, the specific surface area and the pore volume of the drug-loaded carrier are obviously reduced compared with the original blank carrier, and the pore diameter is also obviously reduced, which fully proves that the drug is effectively loaded into the mesoporous pore canal of the carrier.

TABLE 4 variation of specific surface area, pore volume and pore size before and after drug delivery

The drug carrier FTIR results are shown in fig. 19.

The result is analyzed according to the peak position, and compared with the IMC of the bulk drug, after the carrier is loaded with the drug, the drug carrier is a group characteristic peak of the bulk drug with the IMC, so that the IMC is further proved to be successfully loaded into the pore canal.

XRD analysis was performed on a series of samples of IMC, physical mixtures of drug carrier with blank carrier and IMC, and the results are shown in FIG. 22.

The results showed that no crystal diffraction peaks were observed in the pattern for the empty vector and that all exhibited typical broad peaks, indicating that the vector was in an amorphous state. And a large number of sharp diffraction peaks are shown in the IMC map, which indicates that the medicine has a high crystallization structure. The physical mixture of IMC and MSNs, VSNs and CVSNs also exhibited several stronger drug diffraction peaks, compared with imc@msn, imc@vsn, imc@cvsn drug-loaded carriers which showed almost no drug diffraction peaks, indicating that the drug had been transformed into a crystalline form and existed in an amorphous state after being loaded.

DSC analysis was performed on a series of samples such as IMC, physical mixtures of drug carrier with blank carrier and IMC, and the results are shown in FIG. 23. The result shows that the IMC has obvious endothermic peak at about 160 ℃, which indicates that the IMC of the crude drug is in a crystallization state. The physical mixture of IMC and support exhibited relatively small endothermic peaks around 160 ℃, further confirming their crystalline state. While no endothermic peak was observed in the empty vector MSNs, VSNs, CVSNs, indicating that both were in an amorphous state. And after the IMC is loaded into the carrier, there is almost no endothermic peak at 160 ℃, which indicates that the crystal form change occurs after the drug is loaded into the carrier, and the drug exists in an amorphous state.

Example 15

Wettability of drug carrier

The wettability of the drug carrier is shown in figure 24. Fig. 24 shows the trend of the initial contact angle of the wettability (a) and the contact angle (B) of the drug carrier with time, the initial contact angle of the IMC drug substance after the carrier is loaded is obviously reduced, the IMC contact angle of the drug substance is reduced from 66.09 ° to 41.57 ° in 30s experimental time, the imc@msn contact angle is reduced from 35.26 ° to 14.65 °, the imc@vsn contact angle is reduced from 27.28 ° to 5.62 °, and the imc@cvsn contact angle is reduced from 23.75 ° to 0 ° in 20 s. The method shows that the load enters the mesoporous silicon to construct a drug delivery system, so that the wettability of the IMC of the bulk drug can be obviously improved, wherein VSNs and CVSNs depend on the surface roughness advantage, and the wettability of the IMC is improved most strongly.

Example 16

In vitro drug dissolution, the drug in vitro dissolution release results are shown in figure 25. The results show that in three different media of PBS with pH values of 6.5, 6.8 and 7.4, the drug carrier can obviously improve the dissolution rate and dissolution amount of the IMC due to the reduction of the particle size of the IMC in the nano-scale pore canal and the change of the crystallization state of the drug (the change from the crystallization state to the amorphous state). In PBS with pH of 6.5, the dissolution rate of the bulk drug IMC reaches 77% at 120min, the dissolution rate of IMC@MSN reaches 94% at 120min, and the dissolution rate of IMC@VSN and IMC@CVSN reaches 100% at 90min, which is consistent with the wettability result. The VSNs and CVSNs have high interfacial activity, can promote the interaction between the VSNs and the dissolution medium, and promote the VSNs and the CVSNs to enter the inner pore canal of the carrier to realize the dissolution of the medicine. In addition, the accumulated release amount and release speed of the IMC bulk drug and the drug carrier carrying the IMC are increased along with the increase of the pH value.

Example 17

The in vivo pharmacokinetic method of IMC is specific, standard curve, precision and extraction recovery rate. The results show that the peak shapes of the IMC and the internal standard are good, the separation is complete, and endogenous substances in blank plasma cannot influence the measurement of the IMC and the internal standard; the retention time of IMC was 7.94min and the retention time of internal standard was 4.12min. Therefore, the method has good specificity for IMC content determination.

The standard curve and the precision recovery rate are shown in the table equation, wherein y is the concentration of the IMC drug, and x is the peak area ratio of the IMC to the internal standard substance. As shown in the table, the ratio of the IMC to the peak area of the internal standard has good linear relation with the concentration, R ² > 0.99, standard curve meets methodological requirements. The RSD of the daily precision and the daytime precision of each concentration is less than 15 percent, and the precision of the method meets the analysis requirement of biological samples. In addition, the extraction recovery rate of NMS and IMC also meet the biological sample measurement requirement.

TABLE 5 standard curve, accuracy and extraction recovery of IMC in plasma samples

Example 18

The results of the drug carrier pharmacokinetic studies are shown in fig. 26, with the plasma concentration versus time curve, and the pharmacokinetic parameters obtained using DAS software are shown in table 6.

TABLE 6 pharmacokinetic parameters of drug substance groups and carrier formulation groups after oral administration

From the peak reaching time, the peak reaching time of the IMC is obviously shortened after the IMC is loaded into the carrier, and the IMC@MSN, the IMC@VSN and the IMC@CVSN can respectively shorten the peak reaching time of the raw material IMC from 12 hours to 6,4 and 8 hours. From the highest blood concentration (C _max ) It can be seen that the order of highest blood concentration is: under the condition that the IMC@VSN is greater than the IMC@CVSN is greater than the IMC@MSN and the administration dosage is the same, the VSN can obviously improve the highest blood concentration of indomethacin, and the relative bioavailability of the IMC@VSN is about 10 times higher than that of the IMC@MSN and is doubled compared with the IMC@MSN. In addition, the bioavailability of the IMC@CVSN is still slightly improved compared with that of the IMC@VSN due to the existence of chiral response.

Example 19

The in vivo distribution of the medicines in the IMC drug-loaded system is shown in figure 27. For IMC raw medicines, the distribution in the liver is always increased, and the distribution of the other viscera 6h is reduced to different degrees. The rapid metabolism of the drugs distributed to each organ is indicated, and for IMC@MSN, the maximum drug content of each organ appears at the different degrees of metabolism of the occurrence rate of 3 hours and 6 hours. After the medicine is loaded into the carrier, the medicine distribution of each organ is increased relative to the raw medicine. For IMC@VSN, the drug content of each organ is almost increased, and the elimination speed is slow. The maximum medicine content of the IMC@CVSN in each organ is 3 hours, compared with the IMC@VSN, the content of each organ is rapidly increased in 1-3 hours, and thereafter, the organs such as the heart and the like are extremely fast metabolized. Notably, the metabolism of imc@vsn was slow, and the drug content remained high for each organ at 6 hours, with a continuous increase in content, with the highest distribution for each vector in all organs tested at 6 hours compared to the vector.

Example 20

The efficacy of the IMC drug-loaded system, and the trend of the rate of swelling of the feet of the rats over time is shown in FIG. 28. From the graph, after 0.5h of administration, the swelling rate of each group is above 30%, and the foot swelling model is successfully constructed. Severe swelling occurred in the negative saline group, and the extent of foot swelling was reduced in the positive IMC drug substance control group and the drug carrier formulation group. The foot swelling rates of the IMC@MSN, IMC@VSN and IMC@CVSN rats are lower than those of the positive control group. From the foot swelling rate decrease amplitude results, the foot swelling rates of the IMC@VSN and the IMC@CVSN decrease to less than 10% within 3 hours, and still decrease continuously at 3-6 hours compared with the IMC@MSN, and decrease to less than 5% at 6 hours

The morphology of the inflammatory site and the pathological tissue section are shown in fig. 27.

The results show that from the visual ankle swelling morphology, the ankle joint still has more serious swelling at 6h in the normal saline group, and the ankle swelling degree of the other groups is reduced in different degrees due to the anti-inflammatory effect of the medicine. Bleeding and edema of subcutaneous tissues of a physiological saline group, infiltration of a large amount of inflammatory cells (including neutral cells, lymphocytes and the like), subcutaneous and muscle tissue bleeding of an IMC bulk drug group, slight edema and inflammatory cell infiltration, and visible inflammatory cell infiltration of the subcutaneous tissues of an IMC@MSN group, slight edema; while only a small number of inflammatory cells were observed slightly infiltrating in the imc@vsn, imc@cvsn group. The extent of lesions was assessed according to the extent of inflammatory cell infiltration: saline > IMC > IMC@MSN > IMC@VSN > IMC@CVSN.

The embodiment has simple structure and definite drug effect, and the insoluble BCS II non-steroidal drug indomethacin is taken as a model drug, and the mass ratio of IMC to carrier is 1:3, constructing an IMC@MSN, an IMC@VSN and an IMC@CVSN drug carrying system. To study drug loading capacity and in vitro dissolution of drug-loaded carrier, an IMC in vitro analysis method is constructed. Including standard curves for drug concentration and uv absorbance in PBS solutions of methanol, pH 6.5, 6.8 and 7.4.

The drug loading rates of the IMC@MSN, the IMC@VSN and the IMC@CVSN drug loading systems are all about 20%. Compared with the empty carrier without medicine, the specific surface area, the pore volume and the pore diameter of the carrier after medicine loading are obviously reduced. Due to the presence of mesostructure, the drug carrier can change the form in which the drug is present, converting the crystalline drug into an amorphous state. FTIR results show that the peak position of the medicine carrying carrier is matched with that of the blank carrier, the characteristic peak of the IMC bulk medicine is concealed, and the IMC is loaded into the pore canal, so that the medicine dispersion degree is effectively improved. In the XRD analysis results, no crystal diffraction peak was observed in the spectrum and all exhibited typical broad peaks, indicating that the carriers were all in an amorphous state. The medicine shows a large number of sharp diffraction peaks in the IMC map of the crude medicine, namely, the medicine has a high crystallization structure. The physical mixture of the IMC and the blank carrier shows several strong diffraction peaks of the drug, and in contrast, the drug-carrying carriers IMC@MSN, IMC@VSN and IMC@CVSN hardly show diffraction peaks of the drug, which indicate that the drug has been subjected to crystal form transformation after being loaded and exists in an amorphous state. The DSC analysis showed that the drug carrier did not exhibit a distinct endothermic peak compared to the drug substance and physical mixture. The results of XRD and DSC together demonstrate that the drug undergoes a crystal form transformation after being loaded into the pores of the carrier [60]. The carrier has a mesostructure, and the limited nano pore canal size can limit the crystallization of the medicine without crystallization conditions, so that the insoluble medicine is dispersed in the molecular or nano crystal degree.

Compared with the bulk drug, the contact angle of the drug-carrying carrier is obviously reduced, because on one hand, the carrier changes the existence form of the drug, and on the other hand, a large number of hydrophilic hydroxyl groups are distributed on the surface of the carrier and in the pore canal, the capillary effect is generated between the pore canal and the liquid, and the wetting effect of the drug is improved. And the contact angle trend of the IMC@MSN, the IMC@VSN and the IMC@CVSN is consistent with that of the non-drug-carrying carrier.

In an in vitro dissolution experiment, compared with IMC, the drug carrier can obviously improve the release speed and degree of the drug. The drug release speeds of the three drug carriers are basically similar, but the IMC@VSN and the IMC@CVSN still have weak advantages relative to the IMC@MSN, because the nanotube structure on the surface further improves the state of the drug, the size of the drug particles is smaller, and the dispersion state is better.

In experiments in experimental animals, the advantages of VSNs, CVSNs as drug carrier began to appear: the method is characterized in that the method comprises the steps of firstly, carrying out a pharmacokinetics experiment, wherein under the condition that the in-vitro dissolution experiment results of a drug-carrying system are basically similar, the relative bioavailability of IMC@VSN and IMC@CVSN is respectively about 10 times higher than that of IMC, and is doubled compared with that of IMC@MSN. The in vivo drug distribution results show that the in vivo drug distribution of the IMC and the drug-carrying carrier groups in mice is obviously different, particularly the metabolism of IMC@VSN is slower, the drug concentration in each organ is continuously increased for 1-6h, and compared with each carrier in 6h, the carrier has the highest distribution in all organs tested. The pharmacodynamics experimental result is matched with the bioavailability of the pharmacodynamic students, and the final foot swelling rate and the tissue inflammatory trend are IMC > IMC@MSN > IMC@VSN > IMC@CVSN. The reasons are as follows: first, the IMC loaded into the carrier is in an amorphous state, having a higher dissolution rate than the crystalline IMC; second, the possible reason is that the rough outer cluster structure of VSNs can significantly enhance bioadhesion, increase drug absorption, and maintain a high blood concentration for a long time, thereby sufficiently exerting a long-acting effect. The strong delivery advantage of the viral vector depending on the topological structure is initially demonstrated.

The invention successfully synthesizes the chiral virus-like mesoporous silicon modified by L-alanine, has uniform particle size and good dispersibility, and also has excellent wettability and chiral selective adsorption capacity on the basis of retaining the excellent physicochemical properties of the traditional mesoporous silicon, thereby providing reference for the preparation of the mesoporous silicon applied to the field of drug delivery.

In the above examples, the biosafety of the serial silicon-based carriers, including blood compatibility, cytotoxicity and in vivo toxicity after oral administration, was evaluated, and each index meets the basic requirements of biomaterial application. However, there is no hemolytic test for high concentration carrier, and toxicity in vivo should be examined by tail vein injection and the like, and biological safety should be evaluated systematically and comprehensively.

Chiral virucidal mesoporous silicon exhibits excellent intestinal retention and ability to cross intestinal barriers. Based on this result, its application in biomedical fields, such as coating or loading contrast agents like nano gadolinium, should be extended later for enhancing biological imaging.

The chiral virus-like drug delivery system is constructed by taking IMC as a model drug, so that the in-vivo bioavailability of the drug is greatly improved. In view of the longer in vivo residence time, the anti-cancer drugs such as doxorubicin and the like can be loaded subsequently, and a drug slow-release system is constructed.

Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.

Claims

1. A chiral virus-like nano-drug carrier for efficiently overcoming gastrointestinal physiological barriers is characterized in that the carrier is: chiral virus-like mesoporous silicon drug carrier.

2. The method for constructing the chiral virus-like nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract is characterized by comprising the following steps of: indometacin is selected as a model drug, and indometacin IMC is loaded into series of mesoporous silicon spherical mesoporous silicon MSNs, viroid mesoporous silicon VSNs and chiral viroid mesoporous silicon CVSNs.

3. The method for constructing the chiral viromimetic nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract according to claim 2, which is characterized by comprising the following steps: the medicine carrying carrier is prepared by adopting a solvent volatilization method: firstly, preparing an IMC acetone solution with the concentration of 10mg/ml, weighing 60mg of MSN, VSN and CVSN samples, adding the samples into a brown bottle, and then adding 2ml of 10mg/ml indomethacin Xin Bingtong solution to enable the medicine/carrier to be 1/3w/w; sealing, stirring at 300rpm for 24h at room temperature after ultrasonic homogenization, volatilizing the solvent in a vacuum oven at 40 ℃ at an open mouth, and drying to obtain the IMC@MSN, IMC@VSN and IMC@CVSN medicine carrying carrier.

4. The method for constructing the chiral viromimetic nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract according to claim 2, which is characterized in that,

the synthesis method of the MSNs comprises the following steps: sequentially adding 1g of CTAB and 1mL of ammonia water into a solution containing 100mL of deionized water and 30mL of ethanol, dropwise adding 3mL of TEOS after CTAB is completely dissolved, stirring for 4 hours at normal temperature, standing for 24 hours, centrifugally collecting a product, and washing with water and ethanol for multiple times; then, the sample was dried in vacuum at 45℃for 8h. And calcining in a muffle furnace at 550 ℃ for 6 hours to remove CTAB, thus obtaining the MSNs.

5. The method for constructing the chiral viromimetic nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract according to claim 2, which is characterized in that,

the synthesis method of the virus-like mesoporous silicon VSNs comprises the following steps: firstly, adding 40ml of deionized water, 1g of CTAB and 0.18g of TEA into a round-bottom flask, placing the flask in a water bath at 60 ℃ and stirring for 2 hours, and then dropwise adding 20ml of a mixed solution of cyclohexane TEOS, 16ml of cyclohexane and 4ml of TEOS; stirring in water bath at 60deg.C for 48 hr, transferring to oil bath at 98deg.C, stirring for 24 hr, centrifuging, collecting product, and washing with water and ethanol for several times; then, the sample was dried in vacuum at 45℃for 8 hours, and then calcined in a muffle furnace at 550℃for 6 hours to remove CTAB, thereby obtaining VSNs.

6. The method for constructing the chiral viromimetic nano-drug carrier for efficiently overcoming the physiological barrier of the gastrointestinal tract according to claim 2, which is characterized in that,

the synthesis method of chiral virus mesoporous silicon CVSNs comprises the following steps:

synthesis of AVSNs: performing surface amination modification on VSNs by using APTES, weighing 100mg of VSNs with template removed, performing ultrasonic dispersion in absolute ethyl alcohol, adding 300 mu l of APTES, stirring in a water bath at 50 ℃ for 24 hours, washing with absolute ethyl alcohol for multiple times, centrifuging, drying, and collecting samples to obtain amino-functionalized VSNs, namely AVSNs;

removing Boc protecting group by trifluoroacetic acid (TFA), weighing 15mg of Boc-L/D-AVSNs, uniformly suspending in 15ml of DMSO by ultrasound, adding 6.53ml of TFA, reacting at room temperature for 3h, centrifuging, washing with absolute ethyl alcohol, and drying to obtain L/DVSNs, wherein LVSN is CVSN.