阅读说明：本技术 一种多孔分子印迹缓释材料的制备方法 (Preparation method of porous molecularly imprinted sustained-release material ) 是由 陈方方 孙敏 张靖婧 吕成凯 于 2021-01-20 设计创作，主要内容包括：本发明公开了一种多孔分子印迹缓释材料的制备方法,制备过程如下：(1)制备A2(双(2-丙烯酰氧基乙基)二硫酯)单体；(2)制备含二硫键结构的超支化聚合物；(3)沉淀聚合；(4)除去模板分子；(5)得到多孔分子印迹缓释材料。利用双硫键还原敏感断裂的性质,裂解去除超支化聚合物从而在MIPs中形成多孔结构,有利于模板分子与有效结合位点的传质吸附,是一种温和地制备多孔分子印迹材料的化学方法。所制备多孔MIPs具有材料体系的新颖性,所制备材料具有多孔性和选择性吸附性,预计在药物缓释方面具有良好的潜在应用价值。(The invention discloses a preparation method of a porous molecularly imprinted sustained-release material, which comprises the following steps: (1) preparation of A2 (bis (2-acryloyloxyethyl) disulfide) monomer; (2) preparing a hyperbranched polymer containing a disulfide bond structure; (3) precipitation polymerization; (4) removing the template molecule; (5) obtaining the porous molecularly imprinted slow-release material. By utilizing the property of double-sulfur bond reduction sensitive fracture, the hyperbranched polymer is cracked and removed, so that a porous structure is formed in MIPs, mass transfer adsorption of template molecules and effective binding sites is facilitated, and the method is a chemical method for gently preparing the porous molecularly imprinted material. The prepared porous MIPs have the novelty of a material system, and the prepared material has porosity and selective adsorbability and is expected to have good potential application value in the aspect of drug slow release.)

1. A preparation method of a porous molecularly imprinted slow-release material is characterized by comprising the following preparation processes:

(1) preparation of a2 monomer: preparing A2 monomer containing a reversible disulfide bond structure and a terminal double bond by using disulfide containing disulfide and acryloyl chloride containing a terminal double bond;

(2) preparing a hyperbranched polymer containing a disulfide bond structure: adding the A2 monomer synthesized in the step (1) into a B3 monomer, and synthesizing a hyperbranched polymer containing a disulfide bond structure and having a double bond at the tail end through a Michael addition reaction;

(3) precipitation polymerization: taking the hyperbranched polymer synthesized in the step (2) as a reaction substrate, taking thiamine hydrochloride as a template molecule, taking methacrylic acid as a functional monomer, taking ethylene glycol dimethacrylate as a cross-linking agent, taking azobisisobutyronitrile as a thermal initiator, and carrying out precipitation polymerization in acetonitrile to obtain a printed polymer;

(4) removing the template molecule: adding methanol-acetic acid-water solution to wash the imprinted polymer in the step (3) to remove the template molecules;

(5) obtaining the porous molecularly imprinted slow-release material.

2. The preparation method of the porous molecularly imprinted slow-release material of claim 1, wherein before the A2 monomer is prepared, the reaction system is subjected to anhydrous and oxygen-free treatment.

3. The method for preparing the porous molecularly imprinted slow-release material of claim 1, wherein in the step (1), the molar ratio of the A2 to the B3 is 2:1-3: 1.

4. The method for preparing the porous molecularly imprinted slow-release material of claim 1, wherein in the step (2), the A2 and the B3 are reacted in 2-8mL of chloroform at 45 ℃ for 2 days.

5. The preparation method of the porous molecularly imprinted slow-release material according to claim 4, wherein the concentration of the chloroform monomer is 0.4-1.8 mol/L.

6. The preparation method of the porous molecularly imprinted slow-release material according to claim 1, wherein the step (3) is carried out by bubbling for deoxygenation for 15min and reacting for 24h in an oil bath at 60 ℃.

7. The method for preparing a porous molecularly imprinted slow-release material according to claim 1, wherein the step (4) is that after filtration, the material is put into filter paper for sealing, then the material is put into a Soxhlet extractor, the mixed solvent of methanol, water and glacial acetic acid is added, and the material is washed for 3 days at 110 ℃.

8. The preparation method of the porous molecularly imprinted slow-release material according to claim 7, wherein the volume ratio of the mixed solvent of methanol, water and glacial acetic acid is 60:30: 10.

Technical Field

The invention belongs to the field of molecular chemistry, and particularly relates to a preparation method of a porous molecularly imprinted sustained-release material.

Background

Molecularly Imprinted Polymers (MIPs) are mainly prepared by copolymerizing template molecules, functional monomers with structure complementarity and cross-linking agents, are completely matched with the template molecules on a spatial structure and sites, and are generally arranged in an ordered state with structure complementarity through hydrogen bonds, electrostatic interaction, hydrophobic interaction and other non-covalent interactions, so that the molecularly imprinted materials have structure-efficiency predetermination, specific identification and wide applicability. The traditional MIPs are prepared by a bulk polymerization method, the polymer is ground, crushed and screened after imprinting, crosslinking and drying to obtain the MIPs with a certain particle size, and the MIPs are applied after template molecules are removed by elution. Although the bulk polymerization process is relatively simple, the preparation method has the problems of time and labor consumption, low yield, irregular shape of the obtained particles, high dispersion of the particle size and the like due to repeated grinding. Therefore, researchers have been improving the preparation of MIPs, and the methods have been improved from bulk polymerization to suspension polymerization, dispersion polymerization, in-situ polymerization, precipitation polymerization, surface imprinting, and the like. In order to realize better transportation and absorption of the drug in vivo and improve the pertinence of the drug to a target tissue, a polymer drug carrier system draws wide attention, and the polymer material controlled release system can improve the persistence and specificity of the drug and effectively and simply improve the curative effect and the safety of the existing drug. The MIPs can further delay the release of the drugs from the matrix due to the strong hydrogen bonding effect of the template drugs and the functional monomers in the polymer, and have the potential of being drug carriers. As drug carriers, MIPs have important advantages in drug sustained release, selective release of drug enantiomers, and improvement of drug loading of drug carriers.

Aiming at the problems of too deep and too tight embedding of imprinting holes, non-uniform binding sites, poor accessibility, slow recognition kinetics and the like existing in the imprinting technology, the porous-structure MIPs which are beneficial to the removal and recombination of template molecules are synthesized on the basis of the existing preparation method of the molecular imprinting material, and the method has positive effects on improving the number and the utilization rate of effective recognition sites of the MIPs, reducing the embedding of the effective sites and improving the adsorption capacity. A synergistic path of a 'reversible covalent bond' and a 'hyperbranched polymer' is adopted, a hyperbranched polyamidoamine polymer with the tail end of C-C and containing disulfide bonds is polymerized through Michael addition reaction, and imprinting reaction can be directly carried out on the surface of the hyperbranched polyamidoamine polymer through the pore-forming action of reduction sensitive broken disulfide bonds in the preparation process of MIPs (million Instructions Per second) so as to be used as a matrix to prepare porous MIPs (million Instructions Per Mass.), the number and the utilization rate of effective recognition sites are improved, the embedding of the effective sites is reduced, the adsorption capacity is improved, and the selective entrapment and controlled release efficiency of model drug molecules is improved, so that the functions and the efficiency.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a preparation method of a porous molecularly imprinted slow-release material based on a hyperbranched polymer. The technical problem to be solved by the invention is realized by the following technical scheme:

the invention provides a preparation method of a porous molecularly imprinted sustained-release material, which comprises the following steps:

(1) preparation of a2 (bis (2-acryloyloxyethyl) disulfide) monomer: preparing A2 monomer with a terminal double bond and a reversible disulfide bond structure by using bis (2-hydroxyethyl) disulfide with a disulfide bond structure and acryloyl chloride with a terminal double bond;

(2) preparing a hyperbranched polymer containing a disulfide bond structure: adding B3(1- (2-aminoethyl) -piperazine) monomer into the A2 monomer synthesized in the step (1), and synthesizing a hyperbranched polymer containing a disulfide bond structure and having a double bond at the tail end through a Michael addition reaction;

(3) precipitation polymerization: taking the hyperbranched polymer synthesized in the step (2) as a reaction substrate, taking thiamine hydrochloride as a template molecule, taking methacrylic acid as a functional monomer, taking ethylene glycol dimethacrylate as a cross-linking agent, taking azobisisobutyronitrile as a thermal initiator, and carrying out precipitation polymerization in acetonitrile to obtain a printed polymer;

(4) removing the template molecule: adding the imprinted polymer in the step (3) into methanol-acetic acid-water solution for washing to remove the template molecules;

(5) obtaining the porous molecularly imprinted slow-release material.

In one embodiment of the present invention, the reaction system is subjected to an anhydrous and oxygen-free treatment prior to the preparation of the a2 monomer.

In one embodiment of the present invention, in the step (1), the molar ratio of the a2 to the B3 is 2:1 to 3: 1.

In one embodiment of the present invention, in the step (2), the A2 is reacted with the B3 in 2-8mL of chloroform at 45 ℃ for 2 days.

In one embodiment of the invention, the chloroform monomer concentration is 0.4-1.8 mol/L.

In one embodiment of the invention, the (3) process is bubbling deoxygenated for 15min and the reaction is carried out in an oil bath at 60 ℃ for 24 h.

In one embodiment of the present invention, the process (4) is to filter, seal in filter paper, put in a soxhlet extractor, add the mixed solvent of methanol, water and glacial acetic acid, and wash at 110 ℃ for 3 days.

In one embodiment of the invention, the volume ratio of the methanol, water and glacial acetic acid mixed solvent is 60:30: 10.

Compared with the prior art, the invention has the beneficial effects that:

the molecular imprinting technology is a process of preparing a polymer with specific selectivity to a certain target molecule by using the molecule as a template, and has excellent identification, predetermination and practicability. The method can prepare corresponding MIPs according to different targets so as to meet different application requirements. The MIPs have a hydrogen bond function between the template molecule and the functional monomer, and can further delay the release of the drug from the material, so that the MIPs have the potential of being a drug carrier. Hyperbranched polymers are of great interest because of their intramolecular multi-cavity structure, low molecular chain entanglement and good solubility.

Based on the above thought, the invention starts with the preparation of the MIPs with the porous structure, uses the compound containing the reduction-sensitive broken disulfide bond as a monomer to prepare the hyperbranched high polymer, and uses the hyperbranched high polymer as a substrate to perform molecular imprinting, thereby being a new thought for preparing the MIPs with the porous structure. By utilizing the property of double-sulfur bond reduction sensitive fracture, the hyperbranched polymer is cracked and removed, so that a porous structure is formed in MIPs, mass transfer adsorption of template molecules and effective binding sites is facilitated, and the method is a chemical method for gently preparing the porous molecularly imprinted material. The prepared porous MIPs have the novelty of a material system, and the prepared material has porosity and selective adsorbability and is expected to have good potential application value in the aspect of drug slow release.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Drawings

FIG. 1 is a schematic diagram of a porous molecularly imprinted material provided in an embodiment of the invention;

FIG. 2 is a synthetic route of A2 monomer provided by the embodiments of the present invention;

FIG. 3 is a 1H-NMR spectrum of A2 monomer

FIG. 4 is a synthesis scheme of a HBP-AP hyperbranched polymer provided by an embodiment of the present invention

FIG. 5 is a schematic diagram showing the change of GPC curve with time in a reductive degradation process according to an embodiment of the present invention

Fig. 6 is an SEM image of four groups of materials magnified 10000 times provided by an embodiment of the present invention: (a) MIPs-1, (b) NIPs-1, (c) MIPs-2, and (d) NIPs-2.

FIG. 7 shows the drug loading performance of a porous imprinted material provided in an embodiment of the present invention (MIPs-1, NIPs-1 for hyperbranched degradation-based porous imprinted material; MIPs-2, NIPs-2 for conventional methods)

Fig. 8 shows the slow release of four groups of materials in a buffer solution according to an embodiment of the present invention:

(a)pH＝1.7，(b)pH＝7.4。

Detailed Description

In order to further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description will be made on the preparation method of a porous molecularly imprinted sustained release material according to the present invention with reference to the accompanying drawings and the specific embodiments.

The foregoing and other technical matters, features and effects of the present invention will be apparent from the following detailed description of the embodiments, which is to be read in connection with the accompanying drawings. The technical means and effects of the present invention adopted to achieve the predetermined purpose can be more deeply and specifically understood through the description of the specific embodiments, however, the attached drawings are provided for reference and description only and are not used for limiting the technical scheme of the present invention.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that an article or device that comprises a list of elements does not include only those elements but may include other elements not expressly listed. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of additional like elements in the article or device comprising the element.

Example one

Referring to fig. 1, fig. 1 is a schematic diagram of a preparation process of a porous molecularly imprinted material, in this example, a preparation process of a2 monomer Bis (2-acryloyloxyethyl) Disulfide is shown in fig. 2, Bis (2-hydroxyethyl) Disulfide (BHEDS) and Acryloyl Chloride (AC) are used as raw materials, and Triethylamine (Triethylamine, TEA) is used as an acid binding agent, and a reaction is performed in an ice bath at 0 ℃. Since acryloyl chloride is sensitive to water and can be decomposed when meeting water, the inner wall of the reaction vessel is firstly subjected to water removal and oxygen removal.

The specific operation steps are as follows: coating vacuum ester on the bottle mouth and the bottle stopper of a 1000mL Schlenk bottle with a magnetic stirrer, introducing enough argon through a double-row pipe, vacuumizing, baking the bottle wall for 5min by using a heating gun (with the set temperature of 500 ℃), introducing enough argon after the temperature of the bottle wall is reduced to room temperature, circulating the operations of introducing argon, vacuumizing and heating for three times, and ensuring that the reaction bottle is completely anhydrous and anaerobic. Other glassware was also oven dried. A Schlenk flask was placed in an ice bath, and 200mL of anhydrous THF, 7.266g (40mmol) of BHEDS, 24.531g (240mmol) of TEA were sequentially added to the Schlenk flask under an argon atmosphere, and 17.738g (200mmol) of AC was added to 50mL of anhydrous THF, and slowly added dropwise to the Schlenk flask. After the ice bath is completely melted and the temperature is raised to room temperature, the mixture is removed and reacted for 24 hours. The product is then filtered off with suction, THF is removed by means of a rotary evaporator, the product is dissolved in 100mL of dichloromethane, washed three times with 100mL of NaCl solution and 100mL of deionized water in succession, stirred for 30min with the addition of the appropriate amount of anhydrous Na2SO4 and filtered. The filtrate was rotary evaporated to remove methylene chloride, and then purified by column chromatography (silica gel 200-300 mesh, methylene chloride was used as eluent), followed by rotary evaporation to remove methylene chloride, and dried in a vacuum oven at 35 ℃ for three days to obtain 6.12g of an orange viscous liquid. The synthetic route of the A2 monomer is shown in FIG. 1.

The 1H-NMR spectrum of A2 monomer measured by liquid nuclear magnetic resonance is shown in FIG. 3. In the figure, chemical shifts δ are respectively the characteristic peaks corresponding to protons on-CH 2 at 6.34(a), 6.08(b) and 5.79 (c); delta is respectively 4.35(d) and 2.89(e) corresponding to the characteristic peak of the proton on-CH 2-, and the corresponding integrated area ratio at a, b, c, d, e is 2:2:2:1:1, which is consistent with the expected structural formula. Delta is 7.19 corresponding to the peak of solvent CDCl 3.

Example two

Referring to FIG. 4, FIG. 4 is a synthesis scheme of a HBP-AP hyperbranched polymer, which relates to the "A2 + B3" method for preparing a hyperbranched polymer HBP-AP containing disulfide bond structure.

The bis (2-acryloyloxyethyl) disulfide synthesized in the above step is a2 monomer, and 1- (2-aminoethyl) -piperazine (1- (2-aminoethyl) -piperazine, AP) is B3 monomer. Wherein the molar ratio of A2 to B3 is 2:1-3:1, and the A2 monomer is 0.576g (2-3mmol), the B3 monomer is 260. mu.L (1 mmol). The reaction was carried out in 2 to 8mL of chloroform (monomer concentration: 0.4 to 1.8mol/L) at 45 ℃ for 2 days, respectively. And (3) performing rotary evaporation on the product obtained by the reaction to remove the solvent chloroform, dissolving and precipitating in acetone for three times, and performing rotary evaporation to remove the acetone to obtain yellow viscous liquid, thus obtaining the HBP-AP. The synthetic route of HBP-AP hyperbranched polymer is shown in FIG. 3.

And (3) taking Dithiothreitol (DTT) as a reducing agent, and monitoring the degradation process of the hyperbranched polymer HBP-AP through the change of a GPC curve. To ensure sufficient reaction, the mass ratio of the reducing agent to the polymer was set at 4: 1. The specific experimental process is as follows: HBP-AP (50mg), DTT (200mg) and DMF (20mL) were added to a 50mL beaker and stirred, and at a specified time point 1mL of DMF solution was removed, filtered through a 0.22 μm PTFE frit and injected onto a chromatography column. As shown in FIG. 4, the peak appearing between 10 and 30min corresponds to HBP-AP, and the peak around 24min corresponds to degradation products and DTT. Due to the wide molecular weight distribution of the product, the degradation curve is obviously changed when the degradation time is 1 h. The polymer peak appeared later and later with increasing degradation time and decreased continuously, and the peak of degradation product corresponding to around 24min increased continuously. Dissociation experiments show that disulfide bonds in the hyperbranched polymer can be reduced by DTT and cause the degradation of the polymer, and HBP-AP is completely degraded within 12h in the presence of 4 times of excessive DTT, so that the rapid degradation rate is shown.

EXAMPLE III

The present example relates to the preparation of MIPs.

Taking the hyperbranched polymer HBP-AP synthesized in the step 2 as a reaction substrate, taking Thiamine Hydrochloride (THC) molecules as a template, methacrylic acid (MAA) as a functional monomer, Ethylene Glycol Dimethacrylate (EGDMA) as a cross-linking agent, and Azobisisobutyronitrile (AIBN) as a thermal initiator. A50 mL round-bottom flask is respectively added with 34mg (mmol) of THC, 42 mu L (mmol) of MAA, 480 mu L (mmol) of EGDMA, 30mg (mmol) of AIBN, 6.4-16mL of acetonitrile, 1.6-4mL of deionized water and 0-300mg of HBP-AP. Deoxygenation by bubbling for 15min, and reaction in an oil bath at 60 ℃ for 24h for precipitation polymerization. The oil bath was removed and the reaction was stopped. And respectively filtering the imprinted polymers obtained by the reaction, then placing the imprinted polymers into filter paper for sealing, placing the filter paper into a Soxhlet extractor, adding a mixed solvent, and washing the mixture for 3 days at 110 ℃ to remove the template molecule THC. Because template molecules are easier to elute in the presence of protonic acid, the volume ratio of the added methanol, water and glacial acetic acid mixed solvent is methanol: water: glacial acetic acid 60:30:10, solvent was changed once a day during the wash. And after washing, putting the mixture into an oven at 60 ℃ for drying. And (3) washing the non-imprinted molecular reaction product with deionized water after the non-imprinted molecular reaction is finished, filtering the product, and drying the product in a 60 ℃ drying oven. Meanwhile, non-imprinted materials (NIPs) are prepared for comparison of adsorption performance, no template molecule is added in the preparation process, and the rest is the same as the preparation process of the imprinted materials. 70mg of each of the MIPs and NIPs dried and containing the hyperbranched polymer HBP-AP is sampled, 25mL of DTT with the concentration of 0.02g/mL is added to the rest, and the mixture is stirred for 72 hours at room temperature to dissociate the HBP-AP in the material. The material was then rinsed with deionized water and placed in a 60 ℃ oven to dry.

In order to visually understand the micro-morphology structures of MIPs and NIPs, a scanning electron microscope test was performed, and the results are shown in fig. 6. MIPs-1 and NIPs-1 are porous imprinted materials prepared based on degradation of hyperbranched polymers, and the materials have a coarser porous structure as shown in the figure, and MIPs-1(a) has more hole structures due to imprinting sites formed by THC, monomers and cross-linking agents through non-covalent bond acting force and degradation of the hyperbranched polymers. Compared with the MIPs-2 and NIPs-2 without adding hyperbranched polymer, the structure is more regular.

Example four

The embodiment relates to the research on the drug loading performance of porous MIPs.

Weighing 20mg of quantitative porous imprinting material, respectively dispersing in 50mg/L and 3mL of THC solution, carrying out ultrasonic treatment in an ultrasonic cleaner for 5min to form a uniform suspension, and continuously stirring for 24h at room temperature (25 ℃) until adsorption equilibrium is reached. Centrifuging at 4000rpm for 3min, sucking out supernatant, measuring absorbance abs at maximum absorption wavelength with ultraviolet spectrophotometer, and converting into solution concentration at different adsorption time according to the working curve of absorbance and concentration.

FIG. 7 shows the drug loading curves of the four materials against the template molecule THC at room temperature (25 ℃). It can be seen that both groups of MIPs exhibit better drug loading capacity for THC than NIPs. The maximum drug loading of the MIPs-1 to THC reaches 10.16mg/g, which is obviously higher than that of MIPs-2(6.12mg), NIPs-1(3.80mg) and NIPs-2(3.06 mg). After the hyperbranched polymer contained in the MIPs-1 is dissociated, more channels are formed in the material, the opportunity and probability that THC enters the material to form imprinted sites are increased, and therefore the drug loading capacity of the THC is remarkably increased relative to the MIPs-2. And NIPs-1 only forms an internal channel after the hyperbranched polymer is broken because THC is not imprinted, so that the drug loading is basically not increased relative to NIPs-2.

EXAMPLE five

The present example relates to the study of sustained release properties of porous MIPs.

Weighing 200mg of quantitative solid adsorbent, and respectively dispersing the solid adsorbent in a THC solution with a certain initial concentration, wherein the initial concentration of the solution is 300 mg/L. The mixed solution was kept stirring at room temperature (25 ℃) for 48h until adsorption equilibrium was reached. And directly carrying out suction filtration on the solution, directly drying the powder sample, carrying out ultraviolet absorbance test on the clear filtrate, and converting into the total adsorption amount of the material to THC molecules. Weighing 50mg of sample, stirring in the solution, taking out about 3mL of mixed solution at each certain time, centrifuging at 4000rpm for 3min, sucking out supernatant, and measuring absorbance abs at the maximum absorption wavelength by using an ultraviolet spectrophotometer. The moderate temperature adsorption data in the solution is repeated twice under the same condition, and the average value of the obtained data is taken for calculation, and the maximum deviation of the repeated data is usually less than 5%.

Sustained-release behavior studies were performed on MIPs-1, NIPs-1, MIPs-2, and NIPs-2 in an acidic buffer solution at pH 1.7 and a PBS buffer solution at pH 7.4, respectively. The slow release of the four groups of materials in the buffer solution is shown in figure 8. As can be seen from the figure, MIPs-1 and MIPs-2 each released about 10% of the drug load over the first 1h, while NIPs-1 and NIPs-2 each released about 50% and 80%. Within the next 6h, the release of the NIPs-2 and the NIPs-1 is finished, the release speed of the NIPs-1 is higher than that of the NIPs-2, and at the moment, the MIPs-1 and the MIPs-2 release about 40 percent and 55 percent respectively. And then MIPs-1 and MIPs-2 are continuously released, MIPs-2 is completely released within about 24h, and MIPs-1 is completely released within about 30 h. The release rate of MIPs-2 is also faster than MIPs-1. In PBS buffer solution with pH 7.4, the release ratio of the four materials to THC molecules is not high, the release ratio of non-degraded non-imprinted NIPs-2 is the highest, and the release ratio of degraded imprinted MIPs-1 is the lowest. This is because there is substantially no interaction between the solvent molecules of the neutral buffer solution and the template molecules, and the THC released by the material in the solution is substantially molecules that are adsorbed on the surface of the material or that escape from the interior of the material after soaking. MIPs surface adsorb relatively fewer template molecules than NIPs, and therefore release ratios are also relatively small. The MIPs drug-loading performance after adding the hyperbranched polymer and degrading is obviously improved, and the release time of the drug is relatively longer.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.