CN108272768A

CN108272768A - Load the nanoparticle and its microcapsules of human cytokines

Info

Publication number: CN108272768A
Application number: CN201710983761.7A
Authority: CN
Inventors: 何治宇; 陈永明; 刘利新; 毛海泉; 梁锦荣
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2017-10-20
Filing date: 2017-10-20
Publication date: 2018-07-13
Anticipated expiration: 2037-10-20
Also published as: CN108272768B; WO2019076125A1

Abstract

The invention belongs to nanometer pharmaceutical technology fields; it is related to a kind of nanoparticle of load human cytokines, the microcapsules containing the nanoparticle, the method for preparing the nanoparticle or microcapsules; the purposes of pharmaceutical composition containing the nanoparticle or microcapsules and the nanoparticle or microcapsules.

Description

Load the nanoparticle and its microcapsules of human cytokines

Technical field

The invention belongs to nanometer pharmaceutical technology fields, are related to a kind of nanoparticle of load human cytokines, are received containing described The microcapsules of the grain of rice, the method for preparing the nanoparticle or microcapsules, the pharmaceutical composition containing the nanoparticle or microcapsules, And the purposes of the nanoparticle or microcapsules.

Background technology

Insulin is a kind of proteohormone, by the islet β cell in pancreas, participates in adjusting glycometabolism, controls blood Sugar balance.Exogenous insulin is mainly used to treat diabetes.The convenient administration approach of insulin is to be subcutaneously injected, however, long Phase injection can be such that the compliance of patient reduces, and generate many side effects.

Compared to hypodermic injection, the oral administration route of insulin have patient compliance it is good, it is economical, conveniently, safety etc. it is excellent Point.Insulin by oral administration after, can the portal vein enter body circulation, directly participate in metabolism of the liver to glucose, can be effective The secretion pattern and physiological action of simulation endogenous insulin.But insulin have when being administered by oral route it is following urgently Problem to be solved：Firstly, since the acidic environment in stomach, insulin is easy to be degraded under one's belt；Second, insulin is disappearing Inactivation may be degraded by enzymes by changing in road；Finally, due to the high molecular weight and low fat dissolubility of insulin, in intestinal epithelial cell Permeability it is low, cause oral administration biaavailability relatively low.

In the prior art, there is the nanoparticle of the protein medicaments such as load insulin, be used for the mouth of these drugs Clothes delivering.Using nanoparticle as carrier, help to reduce the influence of acidic environment and/or enzyme environment to drug in stomach.But Often there is the problems such as particle diameter distribution is wide, configuration of surface is uneven, bioavilability is not high or drug effect is not high in these nanoparticles.It is special Not, in gastrointestinal system, it is covered with slime layer in enterocyte cell surface, the mucoprotein in slime layer can pass through electrostatic or hydrophobic work It is complexed with nanoparticle, larger aggregation is formed in situ, nanoparticle is anchored in slime layer, affects nanoparticle in mucus Quick random motion in layer, and then hinder osmotic efficiency of the nanoparticle in slime layer.Therefore, the presence of slime layer becomes Limit the bottleneck problem of oral protein medicament delivery efficiency.

Invention content

The present inventor has obtained a kind of nanometer of load human cytokines by in-depth study and performing creative labour Grain.The present inventor is coated with the medicament-carried nano core of positive electricity using electronegative polyanion, and it is negatively charged to form outer layer Polyanion, it is internal be medicament-carried nano core, nanoparticle with core-shell structure.The nanoparticle of the present invention can be by quiet Electric repulsive interaction reduces the interaction of its own and mucoprotein in slime layer, increases infiltration effect of the nanoparticle in slime layer Rate, thus by more drug deliveries to enterocyte.

Therefore, in one aspect, this application provides a kind of nanoparticles, and it includes cores and the poly- the moon being coated in core Ion；The core includes human cytokines, also includes cell-penetrating peptides (CPP).

In certain embodiments, the polyanion be selected from sodium tripolyphosphate, alginic acid, heparin, hyaluronic acid (HA), Hyaluronate, chondroitin sulfate, polyacrylic polymer, polystyrolsulfon acid Type of Collective object or its arbitrary combination.

In certain embodiments, the polyanion is selected from hyaluronic acid, hyaluronate (such as Sodium Hyaluronate) Or combinations thereof.Hyaluronic acid has the characteristics that derive from a wealth of sources, nontoxic, biodegradable, good biocompatibility, is suitable for medicament-carried nano The preparation of grain.Hyaluronic acid can be converted into hyaluronate under alkaline condition.

In certain embodiments, the weight average molecular weight of the polyanion is 4kDa-200kDa (such as 4kDa- 10kDa, 10kDa-50kDa, 50kDa-100kDa, 100kDa-150kDa or 150kDa-200kDa).

In the nanoparticle of the present invention, the human cytokines that the core includes can be hormone, hormone analogs, enzyme, enzyme Inhibitor or antibody.In certain embodiments, the human cytokines are insulin.

In the nanoparticle of the present invention, the CPP that the core includes can play the role of carrier.CPP is considered to have negative It carries pharmaceutical activity molecule and enhances the ability of its transcellular transport.In certain embodiments, the CPP includes that arginine is residual Base.In certain embodiments, the N-terminal of the CPP is arginine residues.In certain embodiments, the CPP is Penetratin.Penetratin is made of from the homeosis domain of the rqikiwfqnrrmkwkk of drosophila 16 amino acid residues, Its amino acid sequence such as SEQ ID NO：Shown in 1.Penetratin can mediate various hydrophobic macromolecular to enter active somatic cell matter The interior integrality without destroying cell membrane.

In certain embodiments, the CPP is by alkyl modified.Using alkylated CPP as carrier, it is easier to be formed Stablize, the nanoparticle of good dispersion.Alkylated CPP can be obtained by the method comprised the steps of：Make saturated fatty acid Carboxyl and CPP on amino (for example, amino of N-terminal) occur condensation reaction, formed amido bond.Preferably, the condensation is anti- It should be in condensing agent (for example, O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid (TBTU)), solvent (for example, N, N- Dimethylformamide (DMF)) and/or alkaline reagent (for example, n,N-diisopropylethylamine (DIEA)) it is existing under the conditions of carry out.

In certain embodiments, the alkyl is C₁₂-C₁₈Alkyl (such as C₁₂Alkyl, C₁₄Alkyl, C₁₆Alkyl or C₁₈Alkane Base).In certain embodiments, the alkyl is straight chained alkyl.

In certain embodiments, the N-terminal of the CPP is by alkyl modified.

In certain embodiments, the CPP is the Penetratin that N-terminal is modified by n-octadecane base.

The nanoparticle of the present invention can be marked by fluorescent chemicals, therefore, in certain embodiments, the nanoparticle It also include the chromophore of fluorescent chemicals or fluorescent chemicals.The fluorescent chemicals include but not limited to fluorescein isothiocynate (FITC), rhodamine isothiocyanates (RITC), 3H- indoles cyanine type dye (such as Cy3, Cy5) or rhodamine (such as rhodamine 6G, Rhodamine 123, rhodamine B).The fluorescent chemicals can mark on polyanion, human cytokines and/or CPP.

The present invention nanoparticle and it includes core can be any shape.In certain embodiments, the nanometer Grain is spherical shape.In certain embodiments, the core is spherical shape.

In certain embodiments, nanoparticle of the invention has narrow grain size and/or uniform particle diameter distribution.Certain In embodiment, the grain size of the nanoparticle be 100nm-900nm (such as 100nm-200nm, 200-300nm, 300-400nm, 400-500nm, 500-600nm, 600-700nm, 700-800nm or 800-900nm).In certain embodiments, the nanometer The polydispersity index (PDI) of grain grain size is 0.05-0.5 (such as 0.05-0.1,0.1-0.2,0.2-0.3,0.3-0.4 or 0.4- 0.5)。

In certain embodiments, the core that nanoparticle of the invention is included has narrow grain size and/or uniform grain Diameter is distributed.In certain embodiments, the grain size of the core be 30nm-500nm (such as 30nm-100nm, 100-200nm, 200-300nm, 300-400nm or 400-500nm).In certain embodiments, the polydispersity index of the grain size of the core (PDI) it is 0.1-0.5 (such as 0.1-0.2,0.2-0.3,0.3-0.4 or 0.4-0.5).

In certain embodiments, nanoparticle surface of the invention is in electronegativity.In certain embodiments, the nanometer The Zeta potential of grain is that (such as -10mV is to -20mV, -20mV to -30mV, -30mV to -40mV or -40mV by -10mV to -50mV To -50mV).

In certain embodiments, nanoparticle of the invention has higher encapsulation rate and/or drugloading rate.In certain implementations In scheme, the encapsulation rate of the nanoparticle be 90%-99% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In certain embodiments, the drugloading rate of the nanoparticle is 50%-90% (such as 50%- 55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85% or 85%-90%).

In the present invention, the encapsulation rate (EE) and drugloading rate (LC) of nanoparticle can be surveyed by the method comprised the steps of ：1) human cytokines of fluorescent marker is used to be made nanoparticle, 2) suspension comprising the nanoparticle is placed in super filter tube In centrifuged, obtain filtrate；3) fluorescence intensity of human cytokines in filtrate is measured, and then calculates the therapeutic egg in filtrate White concentration, to determine the amount of free human cytokines.Encapsulation rate (EE) and drugloading rate (LC) is calculated as follows：

In certain embodiments, in the nanoparticle, human cytokines：Polyanion：The mass ratio of CPP is 1: 0.5- 0.9: 0.1-0.5 (such as 1: 0.5: 0.1-0.5,1: 0.6: 0.1-0.5,1: 0.7: 0.1-0.5,1: 0.8: 0.1-0.5 or 1: 0.9: 0.1-0.5, such as 1: 0.5-0.9: 0.1,1: 0.5-0.9: 0.2,1: 0.5-0.9: 0.3,1: 0.5-0.9: 0.4 or 1: 0.5-0.9∶0.5).Further, in certain embodiments, the quality sum of polyanion and CPP：The matter of human cytokines Amount is than being 1: 1.In certain embodiments, polyanion：CPP：The mass ratio of human cytokines is 0.9: 0.1: 1,0.8: 0.2 : 1,0.7: 0.3: 1,0.6: 0.4: 1 or 0.5: 0.5: 1.

Present invention also provides a kind of microcapsules, the microcapsules include parietal layer, and the present invention being embedded in parietal layer Nanoparticle, the parietal layer includes enteric material or to be mainly made of enteric material.In the present invention, protein medicaments will be loaded Enteric microcapsule is made in nanoparticle, and nanoparticle stability in the gastrointestinal tract, the spy for improving the release of enteron aisle fraction medicine can be improved Anisotropic, improvement pharmacokinetics behavior, to improve bioavilability, the internal pharmacodynamic result of improvement of drug in vivo.

In certain embodiments, the enteric material is selected from cellulose and its derivates, such as hydroxypropyl methylcellulose neighbour Phthalic acid ester (HPMCP), cellulose acetate phthalate (CAP), 1,2,4 benzenetricarboxylic acid cellulose acetate (CAT) etc.. In certain embodiments, the enteric material is HPMCP.

In certain embodiments, the grain size of the microcapsules is 1-10 μm of (such as 1 μm -2 μm, 2 μm -3 μm, 3 μm of -4 μ M, 4 μm -5 μm, 5 μm -6 μm, 6 μm -7 μm, 7 μm -8 μm, 8 μm -9 μm or 9 μm -10 μm).

In certain embodiments, the shape of the microcapsules is spherical shape.

In certain embodiments, the encapsulation rate of the microcapsules be 30%-95% (such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%).

In the present invention, the encapsulation rate (EE) of microcapsules can be measured by the method comprised the steps of：1) fluorescence is used The human cytokines of label are made nanoparticle, 2) it will the nanoparticle is made microcapsules, 3) include the suspension of the microcapsules Liquid is placed in super filter tube and is centrifuged, and obtains filtrate；4) fluorescence intensity of human cytokines in filtrate is measured, and then calculates filtrate In human cytokines concentration, to determine the amount of free human cytokines.Encapsulation rate (EE) is calculated as follows：

In one aspect, this application provides a kind of pharmaceutical compositions, and it includes the nanoparticles or microcapsules of the present invention.

In certain embodiments, described pharmaceutical composition includes for preventing or treating in the nanoparticle or microcapsules The human cytokines disease that can prevent or treat.In certain embodiments, the human cytokines are insulin, institute Pharmaceutical composition is stated for preventing or treating the hyperglycemia in subject.In certain embodiments, the hyperglycemia packet Include stress induction hyperglycemia, diabetes (including type 1 diabetes and diabetes B) and impaired glucose tolerance.

In certain embodiments, described pharmaceutical composition includes nanoparticle or the micro- glue of prevention or treatment effective dose Capsule.In certain embodiments, described pharmaceutical composition includes one or more pharmaceutical carriers.Medicinal load for use in the present invention Body includes but not limited to filler, diluent, adhesive, wetting agent, disintegrant, lubricant, surfactant, preservative, Toner, corrigent, aromatic, effervescent agent, emulsifier, flocculant, deflocculant, bacteriostatic agent, solubilizer.In certain embodiments In, the pharmaceutical carrier is selected from:Ion-exchanger, aluminium oxide, aluminum stearate, lecithin, haemocyanin (such as human serum egg In vain), glycerine, sorbic acid, potassium sorbate, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal state Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, Beeswax, polyethylene-polyoxypropylene block polymer, lanolin and its arbitrary combination.

In certain embodiments, described pharmaceutical composition includes freeze drying protectant.In certain embodiments, the jelly Dry protective agent is alcohols freeze drying protectant, such as xylitol, mannitol or sorbierite.

The pharmaceutical composition of the present invention can be made into various suitable dosage forms, including but not limited to：Peroral dosage form, injection Type (be for example suitable for be subcutaneously injected, the dosage form of intramuscular injection or intravenous injection), inhalant dosage form, mucosa delivery dosage form or part to Pharmaceutically dosage form.In certain embodiments, described pharmaceutical composition is made into peroral dosage form, for example, tablet, capsule, granule, Oral administration solution, oral administration mixed suspension, micropill preparation or micro-tablet.

In one aspect, this application provides the use that the nanoparticle of the present invention or microcapsules are used to prepare pharmaceutical composition On the way, described pharmaceutical composition can prevent for preventing or treating the human cytokines for including in the nanoparticle or microcapsules Or the disease for the treatment of.In certain embodiments, the human cytokines are insulin, and the disease is hyperglycemia.At certain In a little embodiments, the hyperglycemia includes stress induction hyperglycemia, diabetes (including type 1 diabetes and 2 type glycosurias Disease) and impaired glucose tolerance.

In one aspect, include tested to there is this to need this application provides a kind of prevention or the method for the treatment of disease Nanoparticle, microcapsules or the pharmaceutical composition of the person application present invention, the disease are the nanoparticle, microcapsules or pharmaceutical composition The disease that the human cytokines for including in object can prevent or treat.In certain embodiments, the human cytokines are Insulin, the disease are hyperglycemia.In certain embodiments, the hyperglycemia include stress induction hyperglycemia, Diabetes (including type 1 diabetes and diabetes B) and impaired glucose tolerance.

In the embodiment of the application, the subject is preferably mammal, for example, bovid, equid, Caprid, porcine animals, canid, felid, rodent, primate；For example, the subject is People.

In one aspect, it this application provides the method for preparing nanoparticle of the present invention, the described method comprises the following steps：

Step 1：It is the nanoparticle comprising human cytokines and CPP to prepare nanoparticle A, the nanoparticle A；

Step 2：The nanoparticle A that step 1 obtains is coated with using polyanion.

In certain embodiments, the step 1 includes：Make the solution comprising human cytokines and the solution comprising CPP It is mixed.

In certain embodiments, the step 1 further comprises the steps：

Step 1-1：Solution comprising human cytokines and the solution comprising CPP are provided；

Step 1-2：The dress for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided It sets；

Step 1-3：The solution comprising human cytokines and the solution comprising CPP is set to pass through different channel arrival vortex mixed It closes in region, is mixed, obtain the suspension for including nanoparticle A.

In certain embodiments, the solution comprising CPP of step 1-1 have 0.1-0.5mg/mL (such as 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL or 0.5mg/mL) mass concentration.

In certain embodiments, in step 1-1, include the solution of CPP：Including the quality of the solution of human cytokines is dense Degree is than being 0.1-0.5: 1 (such as 0.1: 1,0.2: 1,0.3: 1,0.4: 1 or 0.5: 1).

In certain embodiments, the solution comprising human cytokines is aqueous solution.

In certain embodiments, the solution comprising CPP is aqueous solution.

In the present invention, including the mass concentration of the solution of human cytokines refers to the matter of the human cytokines in the solution Measure concentration.

In the present invention, including the mass concentration of the solution of CPP refers to the mass concentration of the CPP in the solution.

In certain embodiments, the step 1-1 further includes：Use acid solution (such as hydrochloric acid) or alkaline solution (such as sodium hydroxide solution) by the pH of the solution comprising human cytokines adjust to 6.5-7.0 (such as 6.5,6.6, 6.7,6.8,6.9 or 7.0).

In certain embodiments, the device in the step 1-2 is multiple entry vortex mixer (multi-inlet vortex mixer(MIVM))。

In certain embodiments, in the step 1-3, include the solution of human cytokines, and the solution comprising CPP It is at the uniform velocity flowed with identical flow velocity in the channel.In certain embodiments, the flow velocity is 1-50mL/min (such as 1- 15mL/min, 15-25mL/min or 25-50mL/min).

In certain embodiments, the nanoparticle A that prepared by step 1 has narrow grain size and/or uniform particle diameter distribution. In certain embodiments, the grain size of the nanoparticle A is 30nm-500nm (such as 30nm-100nm, 100-200nm, 200- 300nm, 300-400nm or 400-500nm).In certain embodiments, the polydispersity index of the grain size of the nanoparticle A (PDI) it is 0.1-0.5 (such as 0.1-0.2,0.2-0.3,0.3-0.4 or 0.4-0.5).

In certain embodiments, the nanoparticle A that prepared by step 1 is in electropositivity.In certain embodiments, the nanometer The Zeta potential of grain A is that (such as+10mV is to+20mV ,+20mV to+30mV ,+30mV to+40mV or+40mV by+10mV to+50mV To+50mV).

In certain embodiments, the nanoparticle A that prepared by step 1 has higher encapsulation rate and/or drugloading rate.Certain In embodiment, the encapsulation rate of the nanoparticle A be 90%-99% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).In certain embodiments, the drugloading rate of the nanoparticle A be 60%-90% (such as 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85% or 85%-90%).

In the present invention, the encapsulation rate (EE) and drugloading rate (LC) of nanoparticle A can be surveyed by the method comprised the steps of ：1) use the human cytokines of fluorescent marker that nanoparticle A is made；2) suspension comprising the nanoparticle A is placed in ultrafiltration It is centrifuged in pipe, obtains filtrate；3) fluorescence intensity of human cytokines in filtrate is measured, and then is calculated therapeutic in filtrate The concentration of albumen, to determine the amount of free human cytokines.Encapsulation rate (EE) and drugloading rate (LC) is calculated as follows：

In certain embodiments, the step 2 includes：Make the solution comprising polyanion and mixing comprising nanoparticle A Suspension is mixed.

In certain embodiments, the step 2 further comprises the steps：

Step 2-1：Solution comprising polyanion is provided and includes the suspension of nanoparticle A；

Step 2-2：The dress for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided It sets；

Step 2-3：So that the solution comprising polyanion and the suspension comprising nanoparticle A is passed through different channels and reaches whirlpool It flows in Mixed Zone, is mixed, obtain the suspension for including nanoparticle of the present invention.

In certain embodiments, the suspension comprising nanoparticle A in step 2-1 is by including step 1-1, step 1-2 It is obtained with the method for step 1-3.

In certain embodiments, the solution comprising polyanion of step 2-1 have 0.5-0.9mg/mL (such as 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL or 0.9mg/mL) mass concentration.

In certain embodiments, in step 2-1, include the solution of polyanion：Include the matter of the suspension of nanoparticle A Amount concentration ratio is 0.5-0.9: 1 (such as 0.5: 1,0.6: 1,0.7: 1,0.8: 1 or 0.9: 1).

In certain embodiments, the solution comprising polyanion is aqueous solution.

In certain embodiments, in the suspension comprising nanoparticle A, decentralized medium is water.

In the present invention, including the mass concentration of the solution of polyanion refer to the polyanion in the solution quality it is dense Degree.

In the present invention, including the mass concentration of the suspension of nanoparticle A refers to contained by nanoparticle A in the suspension Human cytokines mass concentration.

In certain embodiments, the device in the step 2-2 is multiple entry vortex mixer.

In certain embodiments, in the step 2-3, include the solution of polyanion, and mixed comprising nanoparticle A Suspension at the uniform velocity flows with identical flow velocity in the channel,

.In certain embodiments, the flow velocity be 1-50mL/min (such as 1-15mL/min, 15-25mL/min or 25-50mL/min)。

Present invention also provides the methods for preparing microcapsules of the present invention, the described method comprises the following steps：

Step 1 '：Prepare the nanoparticle of the present invention；

Step 2 '：Using enteric material to step 1 ' obtained nanoparticle is coated with.

In certain embodiments, step 1 ' include：The nanoparticle of the present invention is prepared with preparation method as described above.

In certain embodiments, the step 2 ' further comprise the steps：

Step 2 ' -1：Suspension comprising nanoparticle of the present invention and the solution comprising enteric material are provided；

Step 2 ' -2：The dress for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided It sets；

Step 2 ' -3：Keep the suspension comprising nanoparticle of the present invention, the solution comprising enteric material and optional acidity molten Liquid (such as hydrochloric acid) is reached by different channels in vortex mixing region, is mixed, and is obtained comprising microcapsules of the present invention Solution.

In certain embodiments, the solution comprising enteric material of step 2 ' -1 has 0.25-1mgmL^-1(such as 0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.55mg/mL、0.6mg/mL、 0.65mg/mL, 0.7mg/mL, 0.75mg/mL, 0.8mg/mL, 0.85mg/mL, 0.9mg/mL, 0.95mg/mL or 1mg/mL) Mass concentration.

In certain embodiments, in step 2 ' -1, include the solution of enteric material：Include the suspension of nanoparticle of the present invention The mass concentration ratio of liquid be 0.25-1: 1 (such as 0.25: 1,0.3: 1,0.35: 1,0.4: 1,0.45: 1,0.5: 1,0.55: 1, 0.6: 1,0.65: 1,0.7: 1,0.75: 1,0.8: 1,0.85: 1,0.9: 1,0.95: 1 or 1: 1).

In certain embodiments, the solution comprising enteric material is aqueous solution.

In certain embodiments, in the suspension comprising nanoparticle of the present invention, decentralized medium is water.

In the present invention, including the mass concentration of the solution of enteric material refer to the enteric material in the solution quality it is dense Degree.

In the present invention, including the mass concentration of the suspension of nanoparticle of the present invention refers to the present invention in the suspension The mass concentration of human cytokines contained by nanoparticle.

In certain embodiments, the device in step 2 ' -2 is multiple entry vortex mixer.

In certain embodiments, in step 2 ' -3, include the solution of enteric material, and include nanometer of the present invention The suspension of grain is at the uniform velocity flowed with identical flow velocity in the channel.In certain embodiments, the flow velocity is 1-50mL/min (such as 1-15mL/min, 15-25mL/min or 25-50mL/min).

Fig. 1 illustratively shows the process for preparing nanoparticle and microcapsules of the present invention, wherein is used to prepare nanoparticle Human cytokines are insulin, and polyanion is hyaluronic acid (HA), and the enteric material for being used to prepare microcapsules is HPMCP.System Standby process is as follows：The first step mixes CPP solution with insulin solutions in multiple entry vortex mixer, forms surface Positively charged nanoparticle A (NP-A)；Second step mixes the suspension comprising NP-A with the solution comprising HA, formed with NP-A is core, the nanoparticle (NP-B) of pan coating HA；Third walks, and by the suspension comprising NP-B, includes the solution of HPMCP It is mixed with the pH dilute hydrochloric acid for being 2.5, so that HPMCP is coated on nanoparticle, obtain enteric microcapsule.In each step, using entering more Opening vortex mixer quickly mixes liquid.

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, involved laboratory operation step is to be widely used in corresponding field herein Conventional steps.Meanwhile for a better understanding of the present invention, the definition and explanation of relational language is provided below.

As used in this article, term " human cytokines " refers to the albumen that can be used in preventing or treating disease, including But it is not limited to hormone, hormone analogs, enzyme, enzyme inhibitor and antibody.

As used in this article, term " cell-penetrating peptides " (cell penetrating peptide, CPP) refers to by one As be no more than the small peptides of 30 amino acid constructs, cell can be entered across cell membrane, can be for exogenous molecule be taken Band enters cell.Cell-penetrating peptides can be naturally occurring peptide or artificial synthesized peptide.Common cell-penetrating peptides include：Sun Ion CPP, such as：TAT (48-60), Penetratin, poly arginine, Oct4, WT1-pTj, DPV3；Amphipathic CPP, such as： Transportan、MAP、VP22、Pep1、KW；Hydrophobicity CPP, such as：KFGF、FGF12、Integrinβ3Peptide、 C105Y、TP2.The source of common cell-penetrating peptides and sequence can be found in, such as Joshua D.Ramsey, Nicholas H.Flynn.Cell-penetrating peptides transport therapeutics into cells, Pharmacology&Therapeutics 154(2015)78–86.Cell-penetrating peptides can be modified, for example, in CPP C-terminal or N-terminal modified (such as alkylation modification).

As used in this article, term " saturated fatty acid " refers to the saturated hydrocarbon chains that at least carboxyl is contained in one end, wherein The saturated hydrocarbon chains are mostly straight chain, and carbon atom number can be less than 6,6-12 or more than 12 (such as 12-18).It is full Example with aliphatic acid includes but not limited to：Caproic acid, octanoic acid, capric acid, lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid), Palmitic acid (hexadecanoic acid), stearic acid (octadecanoid acid), arachidic acid (arachic acid).

As used in this article, term " nanoparticle " refers to size (diameter i.e. in the longest dimension of particle) in nanoscale Particle, such as size be 1-100nm, 100-500nm, 500-1000nm or 1000-2000nm particle.

As used in this article, term " grain size " i.e. " equivalent grain size ", refer to when tested particle certain physical characteristic or When physical behavio(u)r and the most close homogenous spheres (or combination) of a certain diameter, just using the diameter of the sphere (or combination) as tested The equivalent grain size (or size distribution) of particle.

As used in this article, term " average grain diameter " refers to, for one by the different particle group of size and shape At practical population, the hypothetical particle faciation ratio being made of uniform spheroidal particle with one, if the grain size overall length of the two It is identical, then claim the average grain diameter of a diameter of practical population of this spheroidal particle.The measurement method of average grain diameter is this field skill Known to art personnel, such as light scattering method；The measuring instrument of average grain diameter includes but not limited to Malvern particle instrument.

As used in this article, term " microcapsules " refers to a kind of solia particle, with parietal layer, and is embedded in parietal layer In content.The substance for constituting parietal layer is usually macromolecule.Microcapsules can be variously-shaped, such as spherical, and diameter is logical Often in the micron-scale or grade.

As used in this article, term " enteric microcapsule " refers to made from the main material using enteric material as parietal layer Microcapsules are resistant to hydrochloric acid in gastric juice, can be disintegrated after entering enteron aisle and release content.

As used in this article, term " enteric material " refers to insoluble in gastric juice or almost insoluble, and the energy in intestinal juice The material of disintegration or dissolving.The solubility of enteric material is different and different with pH.Enteric material for use in the present invention include but It is not limited to cellulose and its derivates, such as hypromellose phthalate (HPMCP), cellulose acetate Plain (CAP), 1,2,4 benzenetricarboxylic acid cellulose acetate (CAT) etc..

As used in this article, term " suspension " refers to the liquid that dispersing solid particles are formed in liquid dispersion medium Dispersion, the liquid dispersion medium include but not limited to water.

As used in this article, term " about " should be readily appreciated by one skilled in the art, and will be with the upper of its place used Hereafter there is a degree of variation.If according to the context of term application, to those skilled in the art, meaning It is not clear, then " about " meaning that deviation is no more than positive and negative the 10% of the certain number value or range.

As used in this article, term " prevention " refers to the generation for preventing or postponing disease.

As used in this article, term " treatment " refers to curing or at least partly preventing the progress of disease, or alleviate disease Symptom.

Advantageous effect of the invention

The present invention has obtained a kind of nanoparticle of load human cytokines, with nucleocapsid, wherein core includes negative The CPP of human cytokines is carried, shell includes polyanion.The nanoparticle of the present invention realizes higher slime layer permeability and intestines Epithelial cell transfer efficiency finally improves the oral administration biaavailability and drug effect of human cytokines.Further, including this hair The microcapsules of bright nanoparticle can protect nanoparticle in hydrochloric acid in gastric juice and quickly release the drug in enteron aisle, drug effect continued smooth.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, still, art technology Personnel will be understood that following drawings and embodiment are merely to illustrate the present invention, rather than to the restriction of the scope of the present invention.According to attached The following detailed description of figure and preferred embodiment, various purposes of the invention and advantageous aspect carry out those skilled in the art It says and will be apparent.

Description of the drawings

Fig. 2 show embodiment 1 prepare nanoparticle during, the initial pH value of flow velocity 1 and CPP solution is to NP-A The influence of particle size, dispersion degree and/or surface potential, data are average value ± SD (n=6) in figure.

Fig. 2A) show that flow velocity 1 is 50mLmin^-1Under conditions of, NP-A made from the CPP solution of different initial pH values Particle size (■), dispersion degree (▲) and surface potential (▼).The result shows that in the range of pH is 5.0~8.0, with The raising of CPP solution initial pH values, the grain size and surface potential of NP-A reduce.

Fig. 2 B) it shows under conditions of CPP solution initial pH values are 8.0, the grain size of 1 time different in flow rate NP-A obtained (■) and dispersion degree (▲).As shown, being 1~50mLmin in flow velocity 1^-1It, can be by NP-A by regulating and controlling flow velocity in range Size controlling within the scope of about 75nm to about 480nm, dispersion degree control in 0.12~0.45 range；And with the increasing of flow velocity Add, the grain size and dispersion degree of NP-A strongly reduce, and increase to 30mLmin in flow velocity^-1After reach a plateau.

Fig. 3 shows that the grain size (■) of NP-B3 and dispersion degree (▲), used NP-A made from 2 times different in flow rate are It is 8.0 in the initial pH of CPP solution, flow velocity 1 is 50mLmin^-1Under conditions of it is obtained.As shown, carrying with flow velocity The grain size of height, NP-B is down to about 105nm from about 219nm, and dispersion degree is down to 0.067 from 0.46.

Fig. 4 shows the pattern of NP-A, NP-B1, NP-B2, NP-B3 in table 1.In figure, all nanoparticles are approximate ball Shape, uniform particle diameter and is uniformly dispersed.It is dyed due to the use of phosphotungstic acid, aobvious dark color position should be the richness of positively charged cell-penetrating peptides Collect region.Scheme the image that A1-A3 is NP-A, scale is respectively 1 μm, 200nm, 100nm.It will be clear that most Cell-penetrating peptides be located at the surface of NP-A, form fluffy thin layer.Figure A4-A6 is respectively the figure of NP-B1, NP-B2, NP-B3 Picture, scale are 200nm.As shown, after HA is coated with, there is visible light gray chromatograph in nanometer core surfaces, show to receive Rice core surfaces, which are successfully coated with, has gone up HA, forms core-shell structure.In addition, after HA is coated with nanometer core surfaces, it is interior The nanometer nuclear structure in portion becomes finer and close, illustrates that the surface to nanometer core is coated with, and can be compacted its internal structure, The structure of entire nanoparticle is set more to stablize.

Fig. 5 shows the test result of FRET experiments.Nanoparticle (Rho123-HA/ (the CPP/ of single fluorescent marker Insulin) and HA/ (CPP/RITC-Insulin)) after 450nm excitations, can respectively at 530nm, 580nm observe To respective emission peak.However, for nanoparticle (Rho123-HA/ (CPP/RITC-Insulin)) prepared by double fluorescence labeling, After the excitation of 450nm, the emission peak (530nm) of Rho123-HA is substantially reduced, and the emission peak of RITC-Insulin (580nm) significantly increases, it is meant that energy is transferred to from donor on receptor, shows that HA is successfully coated on the nanometer for being loaded with insulin In core.

Fig. 6 shows NP-A in table 1 and NP-B3 in low temperature (4 DEG C) or room temperature (25 DEG C), under grain size and grain size point Cloth.Data are average value ± SD (n=6) in figure.NP-A can be stabilized 9h under the conditions of 25 DEG C, and grain size, dispersion degree are bright after 9h It is aobvious to increase.After HA is coated with, the stability of nanoparticle significantly improves, and grain size and dispersion degree are almost unchanged in 48h.Cryogenic conditions Under, the high stability of the nanoparticle of NP-A and NP-B3, grain size, PDI values are without significant changes.The above result shows that nanometer core After hydrophilic HA coatings, stability significantly improves.

Fig. 7 shows NP-A, NP-B1, NP-B2, NP-B3 in simulate the gastric juice (pH2.5) and simulated intestinal fluid (pH7.0) Insulin percentageloss.As shown, NP-A, in simulate the gastric juice (1h) and simulated intestinal fluid (2h), insulin loss amount divides It Gao Da 24% and 21%.After the coating of HA, insulin loss can be substantially reduced.The above results show that the coating of HA can carry The stability of high nanoparticle reduces destruction of the pipe intestinal digesting liquid to nanoparticle, and HA molecular weight is higher, and protecting effect is better.

Fig. 8 shows the pancreas of NP-A and NP-B3 in the PBS containing hyaluronidase (0.01mg/mL) that pH value is 7.4 The insulin releasing of island element release profiles and NP-A and NP-B3 in the PBS without hyaluronidase that pH value is 7.4 is bent Line.As shown, no matter whether there is or not the presence of hyaluronidase, the rate of NP-A release insulin is most fast always, in initial 2h Inside there is apparent phenomenon of burst release (preparation 45%).After HA pan coatings, the reduced rate of insulin is discharged, and And the rate for discharging insulin is reduced with the increase of surface HA molecular weight.

Fig. 9 shows in embodiment 9, after the mucoprotein solution of nanoparticle and various concentration is incubated 1h jointly at 37 DEG C, Insulin content in the aggregation precipitation of formation.By NP-A with 0.5% mucoprotein obtain aggregation precipitation fluorescence it is strong It spends as a contrast and normalizes；# expressions are compared with other 0.5% mucoprotein groups, p ＜ 0.01；* it indicates and other 1.0% viscous eggs White group is compared, p ＜ 0.001.Data are average value ± SD (n=6) in figure.In the presence of no mucoprotein, after each nanoparticle is incubated Insulin content in gained precipitation is extremely low.However, with a concentration of 0.5% or 1.0% mucoprotein be incubated 1h at 37 DEG C after, Severe aggregation has occurred with mucoprotein in NP-A, and the insulin content in centrifugation gained precipitation is high, shows the NP- of the strong positive electricity of surface band A is easily complexed by electrostatic interaction and electronegative mucoprotein into aggregation；After HA pan coatings, the phase of nanoparticle and mucoprotein Interaction obviously weakens, and the amount that aggregation is formed substantially reduces, and with the increase of HA molecular weight, anti-stick protein adsorption effect is more aobvious It writes.After NP-B3 is incubated with a concentration of 0.5% and 1.0% mucoprotein altogether respectively, the amount of insulin in precipitation is only NP-A groups 16.5% and 22.9%.

Figure 10 shows the toxicity of NP-A, NP-B1, NP-B2, NP-B3 to HT29-MTX cells, as shown, nanoparticle To HT29-MTX without apparent cytotoxicity, safety is preferable.

Figure 11 is the Laser Scanning Confocal Microscope photo in embodiment 11, it is shown that free insulin (Figure 11 A), NP-A (figures 11B), the infiltration situation of NP-B1 (Figure 11 C), NP-B2 (Figure 11 D), NP-B3 (Figure 11 E) in slime layer, and it is glutinous penetrating The integrality of nuclear structure during liquid layer.Scale in figure is 10 μm, and green is FITC-CPP, and red is Cy-5- pancreas islet Element, blue are the nucleus of DIPA dyeing.

As shown, for free insulin, after its solution and cell incubation 3h, insulin overall fluorescent intensity is apparent Less than each nanoparticle group, and majority is trapped in slime layer (referring to the photo at 15 μm), can finally slime layer be passed through to reach The amount of cell surface (referring to the photo at 30 μm) is seldom.For NP-A groups, the initial stage that nanoparticle permeates in slime layer is with regard to shape At a large amount of mucoprotein-nanoparticle aggregation, it is difficult to walk and be detained in situ (referring to the photo at 0 μm) downwards, but and pancreas Island element solution is compared, and is increased in the fluorescence intensity for reaching cell level.NP-B is received with the increase of HA molecular weight Hold-up of the grain of rice in slime layer gradually decreases, and the signal for reaching cell surface gradually increases.

In addition, can be clearly seen that from figure, NP-A and NP-B1~3 reach epithelial cell top across slime layer When, the cell-penetrating peptides (green) in nanometer core and insulin (red) signal almost common location illustrate in nanoparticle In top-down process of osmosis, the presence of slime layer has not significant impact the structural intergrity of nanometer core.

Figure 12 shows in embodiment 12 that HT29-MTX cells absorb free insulin and the opposite of each group nanoparticle It measures, in figure, data are average value ± SD (n=6).As shown, the intake of each nanoparticle group is significantly higher than free insulin Group.Nanoparticle being ordered as from low to high according to intake：NP-A ＜ NP-B1 ＜ NP-B2 ＜ NP-B3.The cellular uptake of NP-B3 Highest is measured, is 11 times, 1.9 times of free insulin and NP-A groups respectively.

Figure 13 shows in embodiment 13, and apparent in Caco-2/mucin models of free insulin group and nanoparticle is oozed Saturating constant (Papp).Δ represents p ＜ 0.01；* p ＜ 0.001 are represented.Data are average value ± SD (n=6) in figure.With free pancreas Island element solution is compared, and the Papp values of each nanoparticle group are considerably higher, and the Papp values of NP-B increased compared with NP-A, and with HA The increase of molecular weight, Papp increase, and illustrate that transhipment amount is higher as HA molecular weight is higher.

Figure 14 is the fluorescence co-focusing microphoto of each group mouse jejunum tissue cross-sections in embodiment 14, in figure, is dissociated Insulin and each nanoparticle take on a red color (fluorescence color of Cy-5), and the slime layer in tissue is in green (the fluorescence face of WGA-647 Color), nucleus is in blue (color of DAPI).Scale in figure is 50 μm.

As shown, for oral free insulin solutions group, only observed in the slime layer of intestinal villi and top A small amount of insulin fluorescence (danger signal), it was demonstrated that oral insulin solution is extremely difficult to effective drug absorption.For oral NP-A groups observe that the insulin fluorescence in slime layer is very strong, and the fluorescence in the intestinal villi of lower section is very weak, illustrates more Number NP-A is detained in slime layer, can penetrate slime layer and then the amount absorbed by enterocyte is less.In contrast, NP- It the mucus penetration capacity of B1, NP-B2, NP-B3 and is significantly improved by the efficiency of intestinal epithelial cell absorption and transport.Wherein, NP-B3 Signal in intestinal villi is most strong, and fluorescence multidigit position existing for villus capillary, shows that NP-B3 can be thin by epithelium Born of the same parents effectively absorb and are transported to blood circulation.The above results prove, positively charged relative to surface and unmodified nanometer core, The anti-slime layer of nanoparticle after HA is coated with is detained and quickly the ability of infiltration transhipment downwards significantly increases, and then makes internal pancreas The intestinal absorption efficiency of island element improves.

Figure 15 shows in embodiment 15- experiments 1 that the blood glucose level after the administration of each group rat changes with time.Figure In, # is represented relative to NP-B1 groups, p ＜ 0.05；* it represents relative to NP-B2 groups, p ＜ 0.05.Data are average value ± SD in figure (n=8).As shown, being administered in eight hours, the blood glucose level for taking orally the rat of deionized water is in a slight decrease.Hypodermic injection group Blood glucose level the 25% of initial level is drastically dropped in 1h, blood glucose is all always held at relatively low water in next 4h It is flat.In contrast, it gives notable in the 8h of the blood glucose level of the rat of nanoparticle upon administration but slowly reduces.NP-A、NP- B1 and NP-B2 nanoparticles after oral administration, administration initial stage (0~5h) blood sugar decreasing effect and no significant difference.In 5~8h The blood sugar decreasing effect ratio NP-A and NP-B1 of period, NP-B2 become apparent, and blood glucose value can be reduced to the 45% of initial value.NP- The internal blood sugar decreasing effect of B3 groups is significantly and lasting, and can blood glucose level initial value be slowly smoothly reduced in 8h 40%.

Figure 16 shows in embodiment 15- experiments 2 that the blood glucose level after the administration of each group rat changes with time.Figure In, * is represented relative to NP-B1 microcapsules groups, p ＜ 0.05；# is represented relative to NP-B2 microcapsules groups, p ＜ 0.05.Data in figure For average value ± SD (n=6).

As shown, after oral administration, the blood sugar decreasing effect of NP-B1 and NP-B2 microcapsules groups is apparent.This two groups of rats After oral administration, blood glucose level is continued for smoothly declining, and the blood glucose value of rat is reduced to initially respectively after 8h is administered Horizontal 50% and 40%.The internal blood sugar decreasing effect of NP-B3 microcapsules groups is extremely notable, and rat blood sugar can be made to be put down in 8h It is slow to be enduringly reduced to the 20% of initial value, at this point, the blood glucose level of diabetes rat reaches normal range (NR) (3.5~5mM).

Figure 17 shows in embodiment 16, serum insulin concentration versus time curve in each group rat body.In figure Data are average value ± SD (n=6).Insulin solutions (5IUkg is given as shown, being subcutaneously injected^-1) after, in rat blood serum Insulin concentration sharply increases, and reaches peak value (~120mIUL in 1h^-1).NP-B2 microcapsules and NP-B3 microcapsules are oral After clothes administration, rat blood serum insulin level slowly rises, peak time 4h.It is computed, the micro- glue of NP-B1 microcapsules, NP-B2 The relative bioavailability of capsule and NP-B3 microcapsules is respectively 5.3%, 7.4% and 11%.

Figure 18 shows in embodiment 17, ALP, AST, ALT, γ-GT contents in each group rat blood serum.Data are in figure Average value ± SD (n=6).(model of oral PBS is big with negative control group (normal rat of oral PBS) and positive controls Mouse) it compares, after oral insulin nanoparticle or insulin nanoparticles enteric microcapsule, four kinds of liver drug enzymes in animal pattern body The activity of (ALP, AST, ALT, γ-GT) shows the nanoparticle and its micro- glue of enteric of the oral delivery present invention without apparent increase Capsule is in animal body without overt toxicity.

Figure 19 shows in embodiment 17, the microphoto of each group rat liver tissue HE stained slices, A1：Oral PBS Normal rat；A2：The rat model of oral PBS；A3：The rat model of oral NP-B3；A4：The coated NP- of oral HPMCP The rat model of B3.The case where to control rats, is similar, and the liver structure of experimental group rat is complete, and liver cell arrangement is more whole Together, illustrate that nanoparticle of the invention and its enteric microcapsule after multi-dose repeats to be administered orally, do not cause apparent hepatic injury.

Sequence information

The information of sequence of the present invention is provided in following table：

Sequence number (SEQ ID NO:)	Description
		1	The amino acid sequence of Penetratin

Sequence information

(the SEQ ID NO of sequence 1:1):16aa

RQIKIWFQNRRMKWKK

Specific implementation mode

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.

Material

Fluorescein isothiocynate (FITC), rhodamine isothiocyanates (RITC), Rhodamine 123 (Rho123) are purchased from Shanghai Aladdin biochemical technology Co., Ltd；Cy5 is purchased from Wuhan Little-PA Sciences Co., Ltds；(3- dimethylamino-propyls) Carbodiimide hydrochloride (EDCHCl), n-hydroxysuccinimide (NHS) are purchased from Shanghai Aladdin reagent Co., Ltd； CPP (Ste-RQIKIWFQNRRMKWKK, N-terminal modify the Penetratin of n-octadecane base) has purchased from Nanjing peptide industry biotechnology Limit company, article No.：NJP12879；Mucoprotein, hyaluronidase (HAase) are purchased from Shanghai prophyll biology Co., Ltd；Hyalomitome Sour sodium (Mw is respectively 4.7KDa, 35KDa, 190KDa) is purchased from Shandong Bloomage Freda Biopharm Co., Ltd.；Pork insulin (27.4IU/mg) is purchased from Xuzhou Wan Bang bio-pharmaceuticals Co., Ltd；DAPI, MTT and Alexa647 Wheat germ agglutinin complex (AF-647) is purchased from Abcam companies of Britain；Pork insulin ELISA kit is purchased from Sweden Mercodia companies；Alkaline phosphatase (alkaline phosphatase, ALP) kit, aspartate transaminase (aspartate transaminase, AST) kit, alanine aminotransferase (alanine aminotransferase, ALT) kit, gamma glutamyl transpeptidase (glutamyl transpeptidase, γ-GT) kit are built purchased from Nanjing At bio tech ltd；HPMCP (density=42.0cPs) can capsule Co., Ltd purchased from ShaoXing,ZheJiang city health；Remaining All reagents are commercially available analytical reagents.

Instrument

Malvern laser granulometry (Malvern Zetasizer Nano ZS, Malvern companies of Britain)；Freezing Drying machine (Alpha 1-2LD Plus, German Christ companies)；PH meter (Seven Compact pH Meter, Switzerland Mettler companies)；Automatic dual pure water distiller (Millipore companies of the U.S.)；Supercentrifuge (5810R, German Eppendorf companies)；Ultraviolet-uisible spectrophotometer (Evo lution, Thermo companies of the U.S.)；Fluorescence Sub-ray spectrometer (FS-980, Edinburgh companies of Britain)；Laser confocal microscope (SP8, German Leica companies)；TEM (JEOL-1400, Japanese JEOL companies)；Multi-function microplate reader (Synergy 2, Biotek companies of the U.S.)；Cell incubator (Forma^TMSteri-Cycle^TM, Thermo companies of the U.S.)；Blood glucose meter (UltraVue, the U.S. Johnson companies).

Statistical analysis

All numerical value are represented as average ± standard deviation.Using GraphPad Prism version 5.0 (GraphPad Software, USA) carries out single factor test ANOVA or t test evaluation, and p ＜ 0.05 indicate statistically-significant difference.

The preparation of the coated CPP- insulin nanoparticles of 1 HA of embodiment and enteric microcapsule

1, solution is prepared：Cell-penetrating peptides powder is dissolved in distilled water, a concentration of 0.3mgmL is configured to^-1Solution, It is used in combination the sodium hydroxide solution of 3M to be adjusted to required pH value (5.0,5.5,6.0,6.5,7.0,7.5 or 8.0).Insulin powder is used The dilute hydrochloric acid solution of 0.01M dissolves, and is configured to a concentration of 1mgmL^-1Solution, be used in combination the sodium hydroxide solution of 3M by pH value tune Its pI is crossed, and is slowly adjusted to 6.8, it is made to form transparent clear uniform solution again.The hyaluronic acid of three kinds of different molecular weights Sodium (Mw=4.7KDa, 35KDa or 190KDa) powder with distilled water be prepared into concentration be respectively 0.28,0.30,0.70mgmL^-1 Solution.

2, CPP- insulin nano cores (nanoparticle A) are prepared：By cell-penetrating peptide solution from multiple entry vortex mixer Entrance 2,3 inject, insulin solutions are injected from entrance 1,4, keep the flow velocity of each solution consistent (flow velocity 1), are obtained comprising receiving The suspension of grain of rice A (NP-A).

Prepare the coated CPP- insulin nanoparticles of HA：The obtained suspension containing NP-A is injected from entrance 1,2,3, HA Solution is injected from entrance 4, is kept the flow velocity of each solution consistent (flow velocity 2), suspension is obtained, wherein containing pan coating HA's CPP- insulin nanoparticles (NP-B).It is obtained using the HA (Mw=4.7KDa, 35KDa, 190KDa) of three kinds of different molecular weights Nanoparticle is respectively designated as NP-B1, NP-B2, NP-B3.

3, enteric microcapsule is prepared：Include the suspension of NP-B, entrance 2 from the injection of the entrance 1 of multiple entry vortex mixer Inject ethanol solution (a concentration of 0.75mgmL of HPMCP^-1), entrance 3,4 injects dilute hydrochloric acid (pH 2.5), obtains including enteric The suspension of microcapsules.

Fig. 1 illustratively shows above-mentioned preparation process.

The preparation of the nanoparticle of 2 fluorescent marker of embodiment

1, the synthesis of fluorescent marker component

(1) preparation of the cell-penetrating peptides (FITC-CPP) of marked by fluorescein isothiocyanate

400mg cell-penetrating peptides are dissolved in 40mL 0.05M carbonate buffer solutions (to prepare：1.59g Na₂CO₃、2.93g NaHCO₃Add in 1L distilled waters, pH 9.6) in, 10mg FITC are dissolved in the methanol of 1.5mL, by the methanol solution of FITC and carefully After born of the same parents penetrate peptide solution mixing, it is protected from light for 24 hours under 4 DEG C, gentle agitation.After completion of the reaction, it is with molecular cut off The bag filter of 1.0KDa in deionized water dialyses to sample, and whole process is protected from light, and draws the dialysis medium on the outside of bag filter, prison Its fluorescent value is surveyed, until free dye dialysis finishes.It is kept in dark place after freeze-drying spare.

(2) insulin (the Cy5- pancreases of the insulin (RITC- insulin) of rhodamine isothiocyanates label and Cy5 labels Island element) preparation

400mg insulin is weighed, with 0.05M carbonate buffer solutions (1.59g Na₂CO₃、2.93g NaHCO₃It is bis- to add to 1L Steam water in, pH 9.6) in prepare 5mgmL^-1Insulin solutions, 5mL a concentration of 2mgmL are added^-1RITC methanol it is molten Liquid, in 4 DEG C, be protected from light for 24 hours.In 4 DEG C of dialysis, (molecular cut off of bag filter is 3.5KDa to reaction solution, and dialysis medium is pH 3.0 hydrochloric acid solution).It monitors in dialysis medium without RITC fluorescence signals, you can think that dialysis is thorough, be lyophilized up to RITC- Insulin powder.The labeling method of Cy5- insulin is same as above, the aqueous solution in addition to the methanol solution of RITC to be changed into Cy5.

(3) preparation of the hyaluronic acid (Rho123-HA) of Rhodamine 123 label

The Sodium Hyaluronate for weighing 200mg 190KDa is dissolved in the in the mixed solvent of 20mL methanol and water (1: 1, v/v), fills After dividing dissolving, 95.6mg EDCHCl, 57.4mg NHS are added, adjust pH to 5.0, after reacting 2h, addition 40mg Rhodamine 123s, PH to 6.0 is adjusted, reaction overnight for 24 hours, dialyse in deionized water by the bag filter for being 14KDa with molecular cut off, and timing is inhaled The dialysis medium on the outside of bag filter is taken, fluorescent value is monitored, judges whether free dye dialyses and finish.After dialysis, it is lyophilized into Powder is kept in dark place, spare.

(4) preparation of the hyaluronic acid (RITC-HA) of rhodamine isothiocyanates label

The Sodium Hyaluronate for weighing 200mg 190KDa is dissolved in 20mL methanol and water (1: 1, v/v) in the mixed solvent, fully After dissolving, be added the methanol solution of 40mg RITC, reaction overnight for 24 hours, the bag filter for being 14KDa with molecular cut off go from It dialyses in sub- water, the dialysis medium on the outside of bag filter is drawn in timing, is monitored fluorescent value, is judged whether free dye has dialysed Finish.After dialysis, it is lyophilized into powder, is kept in dark place, it is spare.

2, the process described according to embodiment 1, nanoparticle, preparation condition are prepared using the raw material of fluorescent marker：Cell is worn Saturating peptide solution initial pH value is 8.0, and flow velocity 1 and flow velocity 2 are 50mLmin^-1。

The measurement of 3 grain size of embodiment, particle dispersibility and surface potential

Grain size, dispersion degree and the surface potential of nanoparticle are measured using Malvern laser granulometry.Assay method is such as Under：1) directly the suspension comprising nanoparticle prepared is measured；2) it is 100KDa with molecular cut off by suspension Super filter tube carry out ultrafiltration under the rotating speed of 300 × g, concentrate is resuspended with the phosphate buffer (PBS, pH 6.6) of 0.01M, Subsequent measurements are carried out again.Sample is put into sample cell, after 25 DEG C balance 120s, is measured.Per a sample parallel determination Three times.

Fig. 2A) show that flow velocity 1 is 50mLmin^-1Under conditions of, NP-A made from the CPP solution of different initial pH values Particle size (■), dispersion degree (▲) and surface potential (▼).The result shows that in the range of pH is 5.0~8.0, with The raising of CPP solution initial pH values, the grain size and surface potential of NP-A reduce.The above results show that method of the invention can To obtain the NP-A of different-grain diameter and surface potential by the initial pH value for adjusting CPP solution.

Fig. 2 B) it shows under conditions of CPP solution initial pH values are 8.0, the grain size of 1 time different in flow rate NP-A obtained (■) and dispersion degree (▲).As shown, being 1~50mLmin in flow velocity 1^-1It, can be by NP-A by regulating and controlling flow velocity in range Size controlling within the scope of about 75nm to about 480nm, dispersion degree control in 0.12~0.45 range；And with the increasing of flow velocity Add, the grain size and dispersion degree of NP-A strongly reduce, and increase to 30mLmin in flow velocity^-1After reach a plateau.The above results Show that method of the invention can obtain the NP-A of different-grain diameter and dispersion degree by adjusting flow velocity.

Fig. 3 shows that the grain size (■) of NP-B3 and dispersion degree (▲), used NP-A made from 2 times different in flow rate are It is 8.0 in the initial pH of CPP solution, flow velocity 1 is 50mLmin^-1Under conditions of it is obtained.As shown, carrying with flow velocity The grain size of height, NP-B is down to about 105nm from about 219nm, and dispersion degree is down to 0.067 from 0.46.The above results show of the invention Method can obtain the NP-B of different-grain diameter and dispersion degree by adjusting flow velocity.

It is 8.0 in the initial pH of CPP solution, flow velocity 1 and 2 is 50mLmin^-1Under conditions of, NP-A and NP-B is prepared, Its grain size, particle dispersibility and surface potential are as shown in table 1.

Table 1

As shown in table 1, it is 8.0 in the initial pH of CPP solution, under conditions of flow velocity 1 and 2 is 50mLmin-1, is made NP-A grain size it is small (~75nm), narrowly distributing (PDI:~0.12), surface is positively charged (~+22mV).Through different molecular weight After HA coatings, grain size increases (~100nm), and elecrtonegativity (~-20mV) is presented in surface.The reversion of charge shows that HA is successfully coated with In the surfaces NP-A.After HA is coated with, the particle diameter distribution still more uniformly (PDI of nanoparticle:~0.1).

The measurement of embodiment 4 encapsulation rate and drugloading rate

According to the method for embodiment 2, NP-A is made using RITC- insulin, and further obtain the suspension for including NP-B Liquid.Suspension comprising NP-B is placed in super filter tube (molecular cut off 100KDa), 20min is centrifuged in 4 DEG C, 300 × g. The fluorescence intensity of insulin in filtrate is measured with multi-function microplate reader, and then calculates the insulin concentration in filtrate, is swum with determining From amount of insulin.The encapsulation rate (Encapsulation efficiency, EE) and drugloading rate of nanoparticle is calculated as follows (Loading content, LC)：

Measurement result is as shown in table 2.

Table 2

Sample	HA(M_w)	EE (%)	LC (%)
				NP-A	—	93.9±1.6	75.8±0.3
NP-B1	4.7KDa	95.6±2.4	55.5±0.6
				NP-B2	35KDa	97.3±1.8	66.1±0.4
NP-B3	190KDa	96.6±1.7	66.7±0.5

As shown, the encapsulation rate of each nanoparticle is 90% or more, drugloading rate is 55% or more.In addition, NP-B3 nanometers The total output of grain can reach 6.6gh^-1, equivalent to about 4.5gh^-1Insulin production (load is multiplied by by the total output of nanoparticle Dose is calculated), show that the method for the present invention can be applied to the production of the continuous and scale of insulin nanoparticles.

The observation of 5 nanoparticle microscopic pattern of embodiment

Using the pattern of transmission electron microscope observing nanoparticle.The suspension containing nanoparticle is taken, drops in and is covered with carbon support film On 200 mesh copper mesh, stop 20min, extra liquid is sucked with filter paper, be added dropwise 2% Salkowski's solution dyeing 2min, then with filter Paper sucks extra Salkowski's solution, and naturally dry with transmission electron microscope observing and is taken pictures.

Fig. 4 shows the pattern of NP-A, NP-B1, NP-B2, NP-B3 in table 1.In figure, all nanoparticles are approximate ball Shape, uniform particle diameter and is uniformly dispersed.It is dyed due to the use of phosphotungstic acid, aobvious dark color position should be the richness of positively charged cell-penetrating peptides Collect region.

Scheme the image that A1-A3 is NP-A, scale is respectively 1 μm, 200nm, 100nm.It will be clear that most Cell-penetrating peptides be located at the surface of NP-A, form fluffy thin layer.

Figure A4-A6 is respectively the image of NP-B1, NP-B2, NP-B3, and scale is 200nm.As shown, being coated with through HA Afterwards, there is visible light gray chromatograph in nanometer core surfaces, show that nanometer core surfaces are successfully coated with and gone up HA, form shell Nuclear structure.In addition, after HA is coated with nanometer core surfaces, internal nanometer nuclear structure becomes finer and close, illustrates pair The surface of nanometer core is coated with, its internal structure can be compacted, and the structure of entire nanoparticle is made more to stablize.

The structure of insulin nanoparticles is investigated in embodiment 6 FRET experiments

The present embodiment using fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET) technology confirms the interaction of hyaluronic acid and insulin.

According to the method for embodiment 2,123 (ex of Rho are used：490nm, em：The HA and RITC (ex 530nm) marked： 540nm, em：580nm) the insulin marked prepares the nanoparticle NP-B3 of double fluorescence labeling, forms fluorescence resonance energy pair.With The nanoparticle that the hyaluronic acid of separate marking Rho123 or the insulin of separate marking RITC are prepared as a contrast, The variation of each sample fluorescence spectrum is measured under the excitation wavelength of 450nm.Test results are shown in figure 5.The nanometer of single fluorescent marker Grain (Rho123-HA/ (CPP/Insulin) and HA/ (CPP/RITC-Insulin)) can be distinguished after 450nm excitations Respective emission peak is observed at 530nm, 580nm.However, nanoparticle (the Rho123-HA/ prepared for double fluorescence labeling (CPP/RITC-Insulin)), after the excitation of 450nm, the emission peak (530nm) of Rho123-HA is substantially reduced, and The emission peak (580nm) of RITC-Insulin significantly increases, it is meant that energy is transferred to from donor on receptor, shows that HA is successfully wrapped By on being loaded with the nanometer core of insulin.

The bin stability of 7 nanoparticle of embodiment and the stability in simulating gastro-intestinal Fluid

By in table 1 NP-A and NP-B3 be positioned over low temperature (4 DEG C) or room temperature (25 DEG C), specific time point (1,3,6, 9,12,18,24,48h) monitoring nanoparticle grain size and particle diameter distribution, preliminary assessment nanoparticle is steady during short It is qualitative.

As shown in fig. 6, NP-A can be stabilized 9h under the conditions of 25 DEG C, grain size, dispersion degree obviously increase after 9h.Through HA packets Rear, the stability of nanoparticle significantly improves, and grain size and dispersion degree are almost unchanged in 48h.Under cryogenic conditions, NP-A and NP- The high stability of the nanoparticle of B3, grain size, PDI values are without significant changes.The above result shows that nanometer core is through hydrophilic HA After coating, stability significantly improves.

According to the method for embodiment 2, NP-A, NP-B1, NP-B2 and NP-B3 are prepared using RITC- insulin, is scattered in mould (0.01M PBS, pH 7.0, contains hyaluronidase 0.01mg for quasi- gastric juice (dilute hydrochloric acid solution, pH 2.5) and simulated intestinal fluid mL^-1) in, with super filter tube (molecular cut off 100KDa) ultrafiltration after 1h, 2h, measured in filtrate with multi-function microplate reader Fluorescence intensity (ex：540nm, em：580nm), to calculate insulin content, show that insulin percentageloss, investigation are received Drug loss situation of the grain of rice in simulate the gastric juice and intestinal juice.

8 extracorporeal releasing experiment of embodiment

According to the method for embodiment 2, NP-A and NP-B3 is prepared using RITC- insulin.Nanoparticle is scattered in containing thoroughly Bright matter acid enzyme (0.01mgmL^-1) pH 7.4 PBS in, 37 DEG C, 100rpm constant temperature oscillations.Timing sampling, ultrafiltration (retention point Son amount is 100KDa), the fluorescence intensity (ex of filtrate is measured using multi-function microplate reader：540nm, em：580nm), it and then calculates Go out insulin content, obtains accumulation drug release percentage.Meanwhile nanoparticle is scattered in the dispersion without containing hyaluronidase Release in vitro research is carried out in medium, is used as control experiment.

9 in-vitro simulated mucoprotein of embodiment-nanoparticle absorption

The present embodiment investigates the interaction of nanoparticle and mucoprotein in vitro.According to the method for embodiment 2, use RITC- insulin prepares the nanoparticle of fluorescent marker.The mucoprotein solution and nanoparticle of various concentration are incubated certain time altogether, By monitoring the insulin content in finally formed mucoprotein-nanoparticle aggregation precipitation, to investigate nanoparticle and mucoprotein Interaction situation.

Experimentation：The nanoparticle of fluorescent marker is scattered in the mucoprotein aqueous solution of 0.5% or 1.0% (m/v) respectively In, it is compared with the blank solution without mucoprotein, at 37 DEG C, concussion is incubated 1h.Mixture centrifuges 10min, institute in 3000rpm It must precipitate and be washed 2 times with PBS, the sodium hydroxide solution (3M) of 200 μ L is used in combination to be sufficiently destroyed, vortex oscillation forms uniform solution Afterwards, the fluorescence intensity (ex of solution is measured with multi-function microplate reader：540nm, em：580nm).

Experimental result is as shown in Figure 9.In the presence of no mucoprotein, the insulin after each nanoparticle is incubated in gained precipitation contains It measures extremely low.However, with a concentration of 0.5% or 1.0% mucoprotein be incubated 1h at 37 DEG C after, NP-A and mucoprotein have occurred seriously Assemble, the insulin content in centrifugation gained precipitation is high, shows the NP-A of the strong positive electricity of surface band easily by electrostatic interaction and with negative The mucoprotein of electricity is complexed into aggregation；After HA pan coatings, the interaction of nanoparticle and mucoprotein obviously weakens, aggregation The amount of formation substantially reduces, and with the increase of HA molecular weight, anti-stick protein adsorption effect is more notable.NP-B3 respectively with it is a concentration of After 0.5% and 1.0% mucoprotein is incubated altogether, the amount of insulin in precipitation is only the 16.5% and 22.9% of NP-A groups.

The above results show that the pan coating of HA makes the surface of nanometer core be inverted to presentation negative electricity by strong positive electricity, and make Its hydrophily increases, and then reduces the electrostatic between nanoparticle and mucoprotein and hydrophobic interaction, is expected to increase nanoparticle The top-down osmotic efficiency in slime layer, raising drug-carrying nanometer particle arrival surface epithelial cell and further transmembrane transport Probability.

10 cytotoxicity experiment of embodiment

The present embodiment evaluates safety of each nanoparticle to HT29-MTX cells using MTT detection methods.

The HT29-MTX cells of logarithmic growth phase, with 1.0 × 10⁴The density in a/hole is inoculated in 96 orifice plates, and 200 μ are added L medium cultures suck culture solution afterwards for 24 hours, and trainings of the 200 μ L containing free insulin, NP-A, NP-B1, NP-B2 or NP-B3 is added Base is supported, is incubated for 24 hours in 37 DEG C.20 μ L MTT solution (5mgmL are then added^-1, pH 7.4PBS) continue to be incubated 4h.In removal 100 μ L DMSO are added after clear liquid, shake mixing, then microplate reader are used to measure absorbance in 570nm.It is incubated with blank culture solution Cell, after same treatment as a contrast；Using after acellular culture medium same operation processing as background.Each sample setting three A multiple holes calculate cell survival rate according to the following equation according to measured absorbance：

Wherein, OD_SampleIt is experimental group absorbance, OD_ControlIt is the absorbance of celliferous blank culture solution, OD₀It is background Absorbance.

As shown in Figure 10, each nanoparticle does not show apparent toxicity, safety to the growth of HT29-MTX cells Preferably.

Penetrating power experiment of 11 nanoparticle of embodiment in slime layer

The present embodiment investigates penetrating power of the nanoparticle in slime layer, and nanometer core during penetrating slime layer The integrality of core structure.According to the method for embodiment 2, the nanometer of double fluorescence labeling is prepared using FITC-CPP and Cy5- insulin Grain, and with can HT29-MTX cells slimy be incubated altogether, observed with Laser Scanning Confocal Microscope.

Experimentation：By HT29-MTX cells with 2 × 10⁴The density in a/hole be inoculated in the burnt capsule of dedicated copolymerization (1 × 1cm), it cultivates 2~3 days.The nanoparticle of double fluorescence labeling is added, after being incubated 3h, removes culture medium, PBS is used in combination mildly to wash 2 It is secondary, the sample of slime layer adsorption is removed, cell 10min is fixed with 4% paraformaldehyde, is washed 2 times with PBS.Then altogether It is successively scanned under focusing microscope.As a control group with the free insulin of Cy-5 labels.

Experimental result:Figure 11 shows free insulin (Figure 11 A), NP-A (Figure 11 B), NP-B1 (Figure 11 C), NP-B2 (Figure 11 D), NP-B3 (Figure 11 E) are in the infiltration situation of slime layer and the integrality of nuclear structure.Scale in figure is 10 μm, green Color is FITC-CPP, and red is Cy-5- insulin, and blue is the nucleus of DIPA dyeing.

As shown, for free insulin, after its solution and cell incubation 3h, insulin overall fluorescent intensity is apparent Less than each nanoparticle group, and majority is trapped in slime layer (referring to the photo at 15 μm), can finally slime layer be passed through to reach The amount of cell surface (referring to the photo at 30 μm) is seldom.Reason may is that insulin molecule is directly exposed to mucus environment, It is easily degraded and destroys by the enzyme in mucus, thus the overall fluorescent intensity shown is relatively low.Divide greatly in addition, insulin is hydrophily Son, it is difficult to penetrate through slime layer and be absorbed by enterocyte.

For NP-A groups, the initial stage that nanoparticle permeates in slime layer just forms a large amount of mucoprotein-nanoparticle aggregation, Be difficult to walk and be detained in situ (referring to the photo at 0 μm) downwards, possible reason is that the surfaces NP-A are positively charged, easily with Electrostatic interaction occurs for a variety of mucoproteins in slime layer.But compared with insulin solutions, in the fluorescence intensity for reaching cell level Increase, reason may is that NP-A still has certain protecting effect to the insulin of its inside, and easily with it is negatively charged Cell membrane effect and enter born of the same parents.

For NP-B, with the increase of HA molecular weight, hold-up of the nanoparticle in slime layer gradually decreases, and reaches cell The signal on surface gradually increases, and illustrates that the coating of strong, the electronegative HA of hydrophily improves the stability of nanoparticle really, reduces Compatibility with mucoprotein in slime layer, and protecting effect is related with the molecular weight of HA.

12 HT29-MTX cells of embodiment test the intake of nanoparticle

Experimentation：HT29-MTX cells are with 5 × 10⁴The density in a/hole is inoculated in 24 orifice plates, is added and uses RITC- pancreases The nanoparticle of fluorescent marker is made in island element.After being incubated 3h, culture medium is removed, PBS is used in combination mildly to wash 3 times, removal residual mucus And the sample of cell surface absorption.Using Scepter^TM2.0 hand-held automatic cell counters measure cell number.Then with thin After cellular lysate liquid carries out fully cracking destruction to cell, the fluorescence intensity of cell pyrolysis liquid is measured using multi-function microplate reader.Carefully The relative quantification of born of the same parents' intake is unified for every 5.0 × 10³Fluorescence intensity level corresponding to a cell.The free pancreas islet marked with RITC Element is as a control group.

Experimental result：Figure 12 shows opposite intake of the HT29-MTX cells to free insulin and each group nanoparticle, Data are average value ± SD (n=6) in figure.As shown, the intake of each nanoparticle group is significantly higher than free insulin group.It receives The grain of rice being ordered as from low to high according to intake：NP-A ＜ NP-B1 ＜ NP-B2 ＜ NP-B3.The cellular uptake amount of NP-B3 is most Height is 11 times, 1.9 times of free insulin and NP-A groups respectively.

In the cell model (HT29-MTX) containing slime layer, the intake of nanometer core NP-A is relatively low, it may be possible to by Larger particles easily are gathered into elecrtonegativity mucoprotein in positively charged nanometer core NP-A, the infiltration in slime layer is hindered Hinder, the amount for reaching cell surface substantially reduces, and then cellular uptake efficiency reduces；And the coated nanoparticles of HA can efficiently thoroughly Rete malpighii is crossed, is easy by cellular uptake.

The transepithelial cell traffic of 13 nanoparticle of embodiment

According to the method for embodiment 2, the nanoparticle of fluorescent marker is prepared with RITC- insulin.It is artificially added using surface The Caco-2 cell models of mucoprotein, using the Papp values of insulin as index, investigate nanoparticle transcellular transport ability and Influence of the slime layer to nanoparticle transfer efficiency.

Experimentation：

Cell culture：Caco-2 is placed in 37 DEG C, 5%CO₂Incubator in, respectively with containing 10% fetal calf serum, 1%L- Glutamine, 1% blueness-streptomysin (100IUmL^-1), 1% nonessential amino acid, 10mgmL^-1The DMEM high sugar of mucin Medium culture.

The structure of Caco-2 cell monolayer models：By Caco-2 cell inoculations in 12 orifice plates of Costar Transwell (0.4 μm, diameter 12mm, cellular growth area 1.1cm of polyester membrane aperture on polyester film²), used Millicell every 2 days ERS-2 type resistance instrument monitors its cross-film resistance (Trans-epithellal Electric Resistance, TEER) value；And Replace a culture solution within every two days.After culture 2~3 weeks, when TEER values are higher than 700 Ω cm², you can it is used for subsequent experimental.

Before experiment, the culture solution of the upper and lower rooms Transwell is changed into Hanks balanced salt solutions (HBSS), in 37 DEG C of balances 30min.Upper chamber changes nanoparticle samples of the 200 μ L containing fluorescent marker into, and (wherein insulin is labeled, a concentration of 0.1mgmL^-1) HBSS.After being incubated 4h, lower room solution in right amount is taken, the fluorescence intensity in solution is detected with multi-function microplate reader, to measure pancreas Island element concentration calculates apparent permeability (Papp).As a control group with the free insulin of fluorescent marker.

Q is the drug total amount (ng) of infiltration；C is the initial concentration (ngcm of upper chamber drug^-3)；A is membrane area (cm²)；t It is test period (s).

Experimental result：Figure 13 shows the apparent infiltration of free insulin group and nanoparticle in Caco-2/mucin models Constant (Papp).Δ represents p ＜ 0.01；* p ＜ 0.001 are represented.Data are average value ± SD (n=6) in figure.With free pancreas islet Plain solution is compared, and the Papp values of each nanoparticle group are considerably higher, and the Papp values of NP-B increased compared with NP-A, and with HA points The increase of son amount, Papp increase, and illustrate that transhipment amount is higher as HA molecular weight is higher.

Absorption Study in 14 rat body of embodiment

According to the method for embodiment 2, the nanoparticle of fluorescent marker is prepared with Cy5- insulin, and then tracer insulin is big Absorption behavior in mouse intestinal villi.

Experimentation：

Several normal male SD rats (230~250g) are taken, are divided into 5 groups：Insulin solutions group, NP-A groups, NP-B1 Group, NP-B2 groups, NP-B3 group (insulin doses：1.5mg·kg^-1).To the rat of insulin solutions group, free insulin is given Solution；To each nanoparticle group, by suspension centrifugation (4000rpm) freeze-drying obtained comprising nanoparticle of embodiment 1, by what is obtained Solid is resuspended with distilled water, gives obtained suspension to rat.Before administration, animal overnight fasting can free water.By oral administration After 2h is administered, rat yellow Jackets (0.04mgkg^-1) anaesthetized, rat abdominal cavity is opened along ventrimeson, is gently taken Go out intestinal tissue, and intercepts the jejunal segment (~10cm) close to duodenum end, it is longitudinally cutting along intestinal tube cavity direction, it is light with PBS The soft nanoparticle for rinsing surface adhesion and macroscopic impurity, are then lightly crimped, and liquid nitrogen cooling is quickly put into O.C.T. fixed in (optimum cutting temperature) compound.Histotomy (8 μ are carried out with freezing-microtome M), with the wheat germ agglutinin (WGA) and DAPI dyestuffs of Alexa Fluor647 labels respectively to the mucus of tissue cross-sections Mucoprotein and nucleus in layer are dyed, and confocal microscopy is used in combination.

Staining procedure：

1) slime layer dyeing liquor is prepared：By 1.0mgmL^-1WGA storing solutions diluted with HBSS, the recommendation of WGA solution is dense Degree is 5.0 μ gmL^-1；

2) histotomy removes dyeing liquor after dyeing 10min with dyeing liquor room temperature, and cell is rinsed 2 times with HBSS；

3) after histotomy is protected from light dyeing 5min with nucleus DAPI dye liquors, cell is rinsed 2 times.

Experimental result：

Figure 14 is the fluorescence co-focusing microphoto of each group mouse jejunum tissue cross-sections, in figure, free insulin and each Nanoparticle takes on a red color (fluorescence color of Cy-5), and the slime layer in tissue is in green (fluorescence color of WGA-647), and nucleus is in Blue (color of DAPI).Scale in figure is 50 μm.

As shown, for oral free insulin solutions group, only observed in the slime layer of intestinal villi and top A small amount of insulin fluorescence (danger signal), it was demonstrated that oral insulin solution is extremely difficult to effective drug absorption.For oral NP-A groups observe that the insulin fluorescence in slime layer is very strong, and the fluorescence in the intestinal villi of lower section is very weak, illustrates more Number NP-A is detained in slime layer, can penetrate slime layer and then the amount absorbed by enterocyte is less.This phenomenon and body Outer simulation mucoprotein-nanoparticle adsorption experiment (embodiment 9), penetrating power of the nanoparticle in slime layer test (embodiment 11) and nanoparticle in the cell model (HT29-MTX) containing slime layer intake experiment (embodiment 12) result kissing It closes.In contrast, the mucus penetration capacity of NP-B1, NP-B2, NP-B3 and apparent by the efficiency of intestinal epithelial cell absorption and transport It improves.Wherein, signals of the NP-B3 in intestinal villi is most strong, and fluorescence multidigit position existing for villus capillary, shows NP-B3 can effectively be absorbed by epithelial cell and be transported to blood circulation.The above results prove, it is positively charged relative to surface and without The anti-slime layer of the nanometer core of modification, the nanoparticle after HA is coated with is detained and quickly the ability of infiltration transhipment downwards significantly increases By force, and then the intestinal absorption efficiency of internal insulin is made to improve.

The foundation of 15 type-1 diabetes mellitus rat model of embodiment and hypoglycemic experiment in vivo

The foundation of type-1 diabetes mellitus rat model

Take several male SD rats, intraperitoneal injection streptozotocin (STZ, 70mgkg^-1), injection site is faced upward close to rat The abdomen upper left side of clinostatism.Fasting 6h or more is needed before modeling, restores to give to feed again again after 4h after modeling.Blood glucose is used after a week Instrument measures blood glucose level, if fasting blood glucose level value is higher than 16.0mM, is considered as type-1 diabetes mellitus model modeling success.

Test the hypoglycemic experiment of 1 oral administration nanometer grain

56 type-1 diabetes mellitus rat models are taken, are divided into 7 groups：Negative control group (oral deionized water), insulin solution (insulin concentration 1mgmL^-1) subcutaneous (s.c.) injection group (5IUkg^-1) and insulin solution, NP-A, NP-B1, NP-B2, NP-B3 suspension (insulin concentration 1mgmL^-1) oral medication group (80IUkg^-1).Each nanoparticle suspension is logical Following methods are crossed to obtain：By suspension centrifugation (4000rpm) freeze-drying obtained comprising nanoparticle of embodiment 1, consolidate what is obtained Body is resuspended with distilled water.Before administration, animal overnight fasting can free water.Respectively at administration before and administration after 1,2,3,4,5, 6,7 and 8h tail veins collect blood sample, its blood glucose level is measured with blood glucose meter.

Figure 15 shows that the blood glucose level after the administration of each group rat changes with time.In figure, # is represented relative to NP-B1 Group, p ＜ 0.05；* it represents relative to NP-B2 groups, p ＜ 0.05.Data are average value ± SD (n=8) in figure.

As shown, being administered in eight hours, the blood glucose level for taking orally the rat of deionized water is in a slight decrease, it may be possible to due to Diabetes rat is in fasting state during administration, and there are a basic decreasing value.The blood of the rat of oral insulin solution Sugar level differs unobvious with negative control group.

The blood glucose level of hypodermic injection group drastically drops to the 25% of initial level in 1h, the blood glucose in next 4h All it is always held at reduced levels.But blood glucose level reaches the rapid drawdown effect of reduced levels from higher level within a short period of time It answers, there is high risk for diabetic, it is serious to cause hypoglycemia, stupor, shock or even dead.

In contrast, it gives notable in the 8h of the blood glucose level of the rat of nanoparticle upon administration but slowly reduces.NP- A, NP-B1 and NP-B2 nanoparticles after oral administration, administration initial stage (0~5h) blood sugar decreasing effect and no significant difference. During 5~8h, the blood sugar decreasing effect ratio NP-A and NP-B1 of NP-B2 becomes apparent, and can blood glucose value be reduced to initial value 45%.The internal blood sugar decreasing effect of NP-B3 groups is significantly and lasting, slowly can smoothly be reduced to blood glucose level in 8h just The 40% of initial value.

The above results illustrate that nanoparticle of the invention can smoothly play the role of reducing blood glucose, and drug effect is apparent, safety Property it is high.

Test the hypoglycemic experiment of 2 oral enteric microcapsules

18 type-1 diabetes mellitus rat models are taken, are divided into 3 groups：The coated NP-B1 microcapsules groups of HPMCP, NP-B2 microcapsules Group, NP-B3 microcapsules groups, (insulin concentration 1mgmL^-1) microcapsules are resuspended with distilled water, (80IUkg is administered orally^-1)。

The preparation of microcapsules is to inject FNC entrances 1 by NP-B1, NP-B2, NP-B3 nano-particle that will be prepared, The ethanol solution of the HPMCP prepared injects entrance 2, and entrance 3,4 injects dilute hydrochloric acid solution (pH=2.5), finally obtains corresponding The microcapsules of nano-particle.

Before administration, animal overnight fasting can free water.1,2,3,4,5,6,7 and 8h before administration and after administration Tail vein collects blood sample, and blood glucose level is measured with blood glucose meter.

Figure 16 shows that the blood glucose level after each group Oral Administration in Rats microcapsules changes with time.In figure, * represent relative to NP-B1 microcapsules groups, p ＜ 0.05；# is represented relative to NP-B2 microcapsules groups, p ＜ 0.05.Data are average value ± SD (n in figure =6).

As shown, after oral administration, the blood sugar decreasing effect of NP-B1 and NP-B2 microcapsules groups is apparent.This two groups of rats After oral administration, blood glucose level is continued for smoothly declining, and the blood glucose value of rat is reduced to initially respectively after 8h is administered Horizontal 50% and 40%.The internal blood sugar decreasing effect of NP-B3 microcapsules groups is extremely notable, and rat blood sugar can be made to be put down in 8h It is slow to be enduringly reduced to the 20% of initial value, at this point, the blood glucose level of diabetes rat reaches normal range (NR) (3.5~5mM).Phase For direct subcutaneous insulin injections, microcapsules of the invention slowly, steadily, can be played enduringly hypoglycemic by oral Effect, it is safe, it avoids and generates blood glucose rapid drawdown equivalent risk, can more be received by numerous diabetics.

16 vivo biodistribution availability of embodiment is studied

24 type-1 diabetes mellitus rats are taken, are divided into 4 groups：Insulin solution injection group (s.c.5IUkg^-1), NP-B1 is micro- Capsules group, NP-B2 microcapsules group, NP-B3 microcapsules group (insulin concentration 1mgmL^-1).Microcapsules are resuspended with distilled water, mouth Clothes administration (80IUkg^-1).The preparation of microcapsules is injected by NP-B1, NP-B2, NP-B3 nano-particle that will be prepared The ethanol solution of FNC entrances 1, the HPMCP prepared injects entrance 2, and entrance 3,4 injects dilute hydrochloric acid solution (pH=2.5), finally Obtain the microcapsules of relevant nanometer particle.

0.3,0.6,1,2,3,4,5,6,7 and 8h tail veins collect blood sample after administration, are placed in 1.5mL acquisitions Guan Zhong centrifuges 15min after blood coagulation in 3000rpm, and careful collection upper serum, -80 DEG C save backup, and avoid multigelation.Make With pork insulin ELISA kit the insulin concentration in serum is measured using double solid-phase enzyme immunoassay methods.Concrete operations It is as follows：

Before test, first all reagents and sample are restored to room temperature.

1) prepare enzyme conjugates solution 1 × and cleaning solution 1 ×：By enzyme conjugates solution (Enzyme Conjugate 11 ×) 1: 10 is pressed with enzyme conjugates buffer solution (Enzyme Conjugate Buffer) dilution mixing；Cleaning solution (Wash Buffer 21 ×) it presses 1: 20 and dilutes mixing with deionized water；

2) ELISA Plate in one piece of 96 hole is taken, precision draws the calibration liquid of 25 μ L and blood serum sample is placed in 96 orifice plates, a formula Three parts；

3) per hole be added 100 μ L enzyme conjugates solution 1 ×；

4) (700~900rpm) is vibrated under room temperature (18~25 DEG C) be incubated 2h；

5) board-washing：Overturning ELISA Plate discards reaction solution, and 350 μ L cleaning solutions are added per hole, and jog discards cleaning solution after the several seconds, It is firmly patted on adsorption paper and takes off dry cleaning solution, repeat board-washing 5 times；

6) enzyme bound substrates (Substrate TMB) 200 μ L are added per hole, 15min is incubated under room temperature (18~25 DEG C)；

7) 50 μ L reaction terminating liquids are added per hole, jog 5s is mixed well.Suction is measured under 450nm wavelength with microplate reader Shading value, reading are completed in 30min.

The relative bioavailability (BA) of enteric insulin microcapsules is calculated according to the following formula：

Wherein, AUC_{Oral administration gavage}And AUC_{It is subcutaneously injected}Serum insulin concentration is to the time after respectively representing oral administration gavage and being subcutaneously injected The gross area under curve.

Figure 17 shows serum insulin concentration versus time curve in each group rat body.Data are average value in figure ± SD (n=6).Insulin solutions (5IUkg is given in hypodermic injection^-1) after, insulin concentration sharply increases in rat blood serum, and Reach peak value (~120mIUL in 1h^-1), this is subcutaneously injected isodose with embodiment 15 in diabetes rat model After insulin, blood glucose level is rapidly reduced to the result matches mutually of the 25% of initial value in 1h (referring to Figure 15).NP-B2 is micro- After capsule and NP-B3 microcapsules oral administrations, rat blood serum insulin level slowly rises, peak time 4h.It is computed, The relative bioavailability of NP-B1 microcapsules, NP-B2 microcapsules and NP-B3 microcapsules is respectively 5.3%, 7.4% and 11%.

Safety research in 17 rat body of embodiment

Experimentation：6 normal rats are taken to be used as negative control group, 18 type-1 diabetes mellitus rat models are divided into 3 groups：Mouthful Take PBS groups, oral NP-B3 groups, coated NP-B3 groups (the insulin concentration 1mgmL of oral HPMCP^-1), nanoparticle and microcapsules (80IUkg is resuspended with distilled water^-1).After continuous oral is administered 1 week, tail vein collects blood sample, is placed in 1.5mL acquisitions Guan Zhong, 15min is centrifuged after blood coagulation in 3000rpm, and careful collection upper serum is placed in -80 DEG C and freezes, avoids multigelation.It presses According to the method in kit specification, alkaline phosphatase (ALP), aspartate transaminase (AST), the alanine in serum are measured The content of aminopherase (ALT) and gamma glutamyl transpeptidase (γ-GT).

Animal yellow Jackets (0.04mgkg^-1) anaesthetized, abdominal cavity is opened along ventrimeson, heart is carried out with PBS In perfusion wash body after residual blood, liver organization is gently taken out, after being fixed with 4% paraformaldehyde, through paraffin embedding, use is cold Freeze slicer and carry out histotomy (8 μm), carries out HE dyeing.

Experimental result：Figure 18 shows ALP, AST, ALT, γ-GT contents in each group rat blood serum.Data are flat in figure Mean value ± SD (n=6).With negative control group (normal rat of oral PBS) and positive controls (rat model of oral PBS) It compares, after oral insulin nanoparticle or insulin nanoparticles enteric microcapsule, four kinds of liver drug enzymes in animal pattern body (ALP, AST, ALT, γ-GT) activity without apparent increase, show the oral delivery present invention nanoparticle and its enteric microcapsule moving Without overt toxicity in object.

Figure 19 shows the microphoto of each group rat liver tissue HE stained slices, A1：The normal rat of oral PBS； A2：The rat model of oral PBS；A3：The rat model of oral NP-B3；A4：The model of the coated NP-B3 of oral HPMCP is big Mouse.The case where to control rats, is similar, and the liver structure of experimental group rat is complete, and liver cell arrangement is more neat, illustrates this hair Bright nanoparticle and its enteric microcapsule does not cause apparent hepatic injury after multi-dose repeats to be administered orally.

Although the specific implementation mode of the present invention has obtained detailed description, it will be appreciated by those skilled in the art that：Root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change the guarantor in the present invention Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.

Sequence table

<110>Zhongshan University

<120>Load the nanoparticle and its microcapsules of human cytokines

<130> IDC170050

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Drosophila melanogaster

<400> 1

Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys

1 5 10 15

Claims

1. a kind of nanoparticle, it includes cores and the polyanion being coated in core；The core includes human cytokines, also Including cell-penetrating peptides (CPP)；

Preferably, the polyanion is selected from sodium tripolyphosphate, alginic acid, heparin, hyaluronic acid (HA), hyaluronate, sulphur Aching and limp ossein, polyacrylic polymer, polystyrolsulfon acid Type of Collective object or its arbitrary combination；

Preferably, the polyanion is selected from hyaluronic acid, hyaluronate (such as Sodium Hyaluronate) or combinations thereof；

Preferably, the weight average molecular weight of the polyanion be 4kDa-200kDa (such as 4kDa-10kDa, 10kDa-50kDa, 50kDa-100kDa, 100kDa-150kDa or 150kDa-200kDa)；

Preferably, the human cytokines that the core includes are selected from hormone, hormone analogs, enzyme, enzyme inhibitor or antibody；

Preferably, the human cytokines are insulin；

Preferably, the CPP includes arginine residues；

Preferably, the N-terminal of the CPP is arginine residues；

Preferably, the CPP is Penetratin；

Preferably, the CPP is by alkyl modified；

Preferably, the N-terminal of the CPP is by alkyl modified；

Preferably, the alkyl is C₁₂-C₁₈Alkyl (such as C₁₂Alkyl, C₁₄Alkyl, C₁₆Alkyl or C₁₈Alkyl)；

Preferably, the alkyl is straight chained alkyl；

Preferably, the CPP is the Penetratin that N-terminal is modified by n-octadecane base.

2. the nanoparticle of claim 1 also includes the chromophore of fluorescent chemicals or fluorescent chemicals；

Preferably, the fluorescent chemicals are selected from fluorescein isothiocynate (FITC), rhodamine isothiocyanates (RITC), 3H- Indoles cyanine type dye (such as Cy3, Cy5) or rhodamine (such as rhodamine 6G, Rhodamine 123, rhodamine B)；

Preferably, the fluorescent compound label is on polyanion, human cytokines and/or CPP.

3. the nanoparticle of claims 1 or 2, for spherical shape；

Preferably, the core that the nanoparticle includes is spherical shape；

Preferably, the grain size of the nanoparticle be 100nm-900nm (such as 100nm-200nm, 200-300nm, 300-400nm, 400-500nm, 500-600nm, 600-700nm, 700-800nm or 800-900nm)；

Preferably, the polydispersity index (PDI) of the nanoparticle grain size be 0.05-0.5 (such as 0.05-0.1,0.1-0.2, 0.2-0.3,0.3-0.4 or 0.4-0.5)；

Preferably, the grain size of the core is 30nm-500nm (such as 30nm-100nm, 100-200nm, 200-300nm, 300- 400nm or 400-500nm)；

Preferably, the polydispersity index (PDI) of the grain size of the core is 0.1-0.5 (such as 0.1-0.2,0.2-0.3,0.3- 0.4 or 0.4-0.5)；

Preferably, the Zeta potential of the nanoparticle be -10mV to -50mV (such as -10mV to -20mV, -20mV to -30mV, - 30mV to -40mV or -40mV to -50mV)；

Preferably, the encapsulation rate of the nanoparticle be 90%-99% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%)；

Preferably, the drugloading rate of the nanoparticle be 50%-90% (such as 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85% or 85%-90%).

Preferably, in the nanoparticle, human cytokines：Polyanion：The mass ratio of CPP is 1：0.5-0.9：0.1-0.5 (examples Such as 1：0.5：0.1-0.5、1：0.6：0.1-0.5、1：0.7：0.1-0.5、1：0.8：0.1-0.5 or 1：0.9：0.1-0.5, such as 1：0.5-0.9：0.1、1：0.5-0.9：0.2、1：0.5-0.9：0.3、1：0.5-0.9：0.4 or 1：0.5-0.9：0.5).

4. a kind of microcapsules, the microcapsules include parietal layer, and any one of claim 1-3 being embedded in parietal layer nanometer Grain, the parietal layer include enteric material or are mainly made of enteric material；

Preferably, the enteric material is selected from cellulose and its derivates, such as hypromellose phthalate (HPMCP), cellulose acetate phthalate (CAP), 1,2,4 benzenetricarboxylic acid cellulose acetate (CAT)；

Preferably, the enteric material is HPMCP；

Preferably, the grain size of the microcapsules is 1-10 μm (such as 1 μm -2 μm, 2 μm -3 μm, 3 μm -4 μm, 4 μm -5 μm, 5 μm -6 μm, 6 μm -7 μm, 7 μm -8 μm, 8 μm -9 μm or 9 μm -10 μm)；

Preferably, the shape of the microcapsules is spherical shape；

Preferably, the encapsulation rate of the microcapsules be 30%-95% (such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%).

5. a kind of pharmaceutical composition, it includes the nanoparticle of any one of claim 1-3 or the microcapsules of claim 4；

Preferably, described pharmaceutical composition is for preventing or treating the human cytokines institute for including in the nanoparticle or microcapsules The disease that can prevent or treat；

Preferably, the human cytokines are insulin, and described pharmaceutical composition is for preventing or treating the high blood in subject Sugared disease；

Preferably, the hyperglycemia includes stress induction hyperglycemia, diabetes (including type 1 diabetes and diabetes B) And impaired glucose tolerance；

Preferably, described pharmaceutical composition includes the nanoparticle or microcapsules of prevention or treatment effective dose；

Preferably, described pharmaceutical composition includes one or more pharmaceutical carriers；

Preferably, described pharmaceutical composition includes freeze drying protectant；

Preferably, the freeze drying protectant is alcohols freeze drying protectant, such as xylitol, mannitol or sorbierite；

Preferably, the subject is mammal, such as bovid, equid, caprid, porcine animals, Canidae Animal, felid, rodent, primate；For example, described, subject is a human.

6. the nanoparticle of any one of claim 1-3 or the microcapsules of claim 4 are used to prepare the purposes of pharmaceutical composition, institute Stating pharmaceutical composition can prevent or control for preventing or treating the human cytokines for including in the nanoparticle or microcapsules The disease for the treatment of；

Preferably, the human cytokines are insulin, and the disease is the hyperglycemia in subject；

7. a kind of method of prevention or treatment disease includes applying any one of claim 1-3's to subject in need The pharmaceutical composition of nanoparticle, the microcapsules of claim 4 or claim 5, the disease be the nanoparticle, microcapsules or The disease that the human cytokines for including in pharmaceutical composition can prevent or treat；

Preferably, the human cytokines are insulin, and the disease is hyperglycemia；

8. the method for preparing the nanoparticle of any one of claim 1-3, the described method comprises the following steps：

Step 2：The nanoparticle A that step 1 obtains is coated with using polyanion；

Preferably, the step 1 includes：The solution comprising human cytokines is set to be mixed with the solution comprising CPP；

Preferably, the step 1 further comprises the steps：

Step 1-2：The device for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided；

Step 1-3：The solution comprising human cytokines and the solution comprising CPP is set to reach vortex mixing area by different channels It in domain, is mixed, obtains the suspension for including nanoparticle A；

Preferably, the grain size for the nanoparticle A that prepared by step 1 is 30nm-500nm (such as 30nm-100nm, 100-200nm, 200- 300nm, 300-400nm or 400-500nm)；

Preferably, step 1 prepare nanoparticle A grain size polydispersity index (PDI) be 0.1-0.5 (such as 0.1-0.2, 0.2-0.3,0.3-0.4 or 0.4-0.5)；

Preferably, the Zeta potential for the nanoparticle A that prepared by step 1 is that (such as+10mV is to+20mV ,+20mV by+10mV to+50mV To+30mV ,+30mV to+40mV or+40mV to+50mV)；

Preferably, step 1 prepare nanoparticle A encapsulation rate be 90%-99% (such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%)；

Preferably, step 1 prepare nanoparticle A drugloading rate be 60%-90% (such as 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85% or 85%-90%)；

Preferably, the step 2 includes：The solution comprising polyanion is set to be mixed with the suspension comprising nanoparticle A；

Preferably, the step 2 further comprises the steps：

Step 2-2：The device for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided；

Step 2-3：The solution comprising polyanion and the suspension comprising nanoparticle A is set to pass through different channel arrival vortex mixed It closes in region, is mixed, obtain the suspension for including the nanoparticle；

Preferably, the suspension comprising nanoparticle A in step 2-1 is by the method comprising step 1-1, step 1-2 and step 1-3 It obtains.

9. the method for preparing the microcapsules of claim 4, the described method comprises the following steps：

Step 1 '：Prepare the nanoparticle of any one of claim 1-3；

Step 2 '：Using enteric material to step 1 ' obtained nanoparticle is coated with；

Preferably, step 1 ' include：The nanoparticle of any one of claim to a method 1-3 with claim 8；

Preferably, the step 2 ' further comprise the steps：

Step 2 ' -1：The suspension of nanoparticle comprising any one of claim 1-3 and the solution comprising enteric material are provided；

Step 2 ' -2：The device for including vortex mixing region and multiple channels that vortex mixing region is flowed to for fluid is provided；

Step 2 ' -3：Make the suspension of the nanoparticle comprising any one of claim 1-3, the solution comprising enteric material and optionally Acid solution (such as hydrochloric acid) reached in vortex mixing region by different channel, mixed, obtained comprising described micro- The solution of capsule.