METHOD USING POLYETHYLENE GLYCOL TO PREPARE FIBROIN NANO/MICROSPHERES, AND APPLICATION OF METHOD IN CONTROLLED DRUG RELEASE
20170340575 · 2017-11-30
Assignee
Inventors
Cpc classification
A61K9/19
HUMAN NECESSITIES
A61K47/42
HUMAN NECESSITIES
A61K9/5146
HUMAN NECESSITIES
International classification
Abstract
A method using polyethylene glycol to prepare fibroin nano/microspheres. A fibroin solution having a mass percentage of 1-30% and a polyethylene glycol solution having a mass percentage of 10-60% are first placed in a 4-60° C. environment for 30 minutes, the fibroin protein solution and the polyethylene glycol solution are then mixed, and fibroin nano/microspheres are produced via incubation and centrifugal washing.
Claims
1. A method for preparation of fibroin nano/microspheres using polyethylene glycol, the method comprising: subjecting a 1-30 wt % fibroin solution and a 10-60 wt % polyethylene glycol solution to a temperature of 4-60° C. for 30 min; mixing the fibroin solution and the polyethylene glycol solution at a certain ratio; incubating the mixture for a certain period of time; and subjecting the mixture to centrifugal washing to produce a suspension of fibroin nano/microspheres.
2. A method for the preparation of drug-loaded fibroin nano/microspheres using polyethylene glycol for the controlled release of drugs, the method comprising: dissolving the drug in a solution, wherein the solution comprises a 10-60 wt % polyethylene glycol solution or a 1-30 wt % fibroin solution; mixing the solution with the other of the 10-60 wt % polyethylene glycol solution or the 1-30 wt % fibroin solution to produce a blending solution; incubating the blending solution for a certain period of time; and subjecting the blending solution to centrifugal washing to produce drug-loaded fibroin nano/microspheres.
3. The method according to claim 2, wherein when the drug is hydrophobic, the method comprises dissolving the drug in the polyethylene glycol solution and mixing the polyethylene glycol solution with the fibroin solution to produce the blending solution; and wherein when the drug is not hydrophobic, the method comprises dissolving the drug in the fibroin solution and mixing the fibroin solution with the polyethylene glycol solution to produce the blending solution.
4. The method according to claim 3, wherein the hydrophobic drug comprises curcumin, and wherein the drug that is not hydrophobic comprises hydrophilic drug adriamycin, polypeptide drug octreotide, protein drug bovine serum albumin, macromolecular hydrophilic model drug glucan or fluorescent labeled CdTe quantum dot.
5. The method according to claim 2, wherein the mass ratio of fibroin contained in said fibroin solution to said drug is in the range of 1000:1 to 1:10.
6. The method according to claim 1, wherein the molecular weight of polyethylene glycol is in the range of 2000-20000.
7. The method according to claim 6, wherein when the molecular weight of polyethylene glycol is between 4000 and 6000, the mass percent of polyethylene glycol is 30-60%; when the molecular weight of polyethylene glycol is 10000, the mass percent of polyethylene glycol is 20-50%; and when the molecular weight of polyethylene glycol is 20000, the mass percent of polyethylene glycol is 20-40%.
8. The method according to claim 1, wherein the pH value of said fibroin solution is in the range of 3.0-11.0.
9. The method according to claim 1, wherein the salt ions concentration for treatment of fibroin solution is in the range of 1 mol/L to 0.01 mol/L.
10. The method according to claim 1, wherein the volume ratio of fibroin solution to polyethylene glycol solution is in the range of 5:1 to 1:10.
11. The method according to claim 1, wherein said blending solution is incubated at a temperature from room temperature to 60° C. for 0.5-24 h.
12. The method according to claim 1, wherein the dilution range of said fibroin solution is 5-20 wt %.
13. The method according to claim 1, further comprising: soaking degummed, boiled-off silk in a 9.3 M solution of LiBr and placing the soaked silk in an oven at 60° C. for 4 h to dissolve the silk in order to obtain the fibroin solution; pouring said fibroin solution into a dialysis bag and dialyzing the fibroin solution against deionized water for 3 days; centrifuging the fibroin solution to remove insoluble impurities; and pouring the fibroin solution into a dialysis bag and dialyzing the fibroin solution against 15 wt % solution of polyethylene glycol having a molecular weight of 20000 for 24 h, to obtain the 1-30 wt % fibroin solution.
14. The method according to claim 2, wherein the molecular weight of polyethylene glycol is in the range of 2000-20000.
15. The method according to claim 14, wherein when the molecular weight of polyethylene glycol is from 4000 to 6000, the mass percent of the polyethylene glycol is 30-60%; when the molecular weight of polyethylene glycol is 10000, the mass percent of the polyethylene glycol is 20-50%; and when the molecular weight of polyethylene glycol is 20000, its mass percent is 20-40%.
16. The method according to claim 2, wherein the pH value of said fibroin solution is in the range of 3.0-11.0.
17. The method according to claim 2, wherein the salt ions concentration for treatment of fibroin solution is in the range of 1 mol/L to 0.01 mol/L.
18. The method according to claim 2, wherein the volume ratio of fibroin solution to polyethylene glycol solution is in the range of 5:1 to 1:10.
19. The method according to claim 2, wherein said blending solution is incubated at a temperature from room temperature to 60° C. for 0.5-24 h.
20. The method according to claim 2, wherein the dilution range of said fibroin solution is 5-20 wt %.
21. The method according to claim 2, further comprising: soaking degummed, boiled-off silk is soaked in a 9.3 M solution of LiBr and placing the soaked silk in an oven at 60° C. for 4 h to dissolve the silk in order to obtain the fibroin solution; pouring said fibroin solution into a dialysis bag and dialyzing the fibroin solution against deionized water for 3 days; centrifuging the fibroin solution to remove insoluble impurities; and pouring the fibroin solution into a dialysis bag and dialyzing the fibroin solution against 15 wt % solution of polyethylene glycol having a molecular weight of 20000 for 24 h, to obtain the 1-30 wt. fibroin solution.
Description
DESCRIPTION OF FIGURES
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EMBODIMENTS
[0089] The technical solution in examples of the present invention will be clearly and integrally illustrated hereinafter, and obviously, the described examples are only a part but not all of the examples in the present invention, and these examples are not intended to limit the scope of the present invention. On the basis of examples of the present invention, all other examples acquired by those skilled in the art without any creative works are within the protection scope of the present invention.
[0090] In order to maintain the stability and molecular activity of drug delivery, for the method for preparation of drug-loaded fibroin nano/microparticles used for controlled release of drugs according to the present invention, it is necessary to avoid the use of organic solvents and severe particles/spheres forming conditions in the preparation process. Comparing with current methods of preparing nano/microparticles by phase separation, the particle size and the morphology of microspheres prepared by phase separation can be regulated by changing the molecular weight of PVA (polyvinyl alcohol) as well as the weight ratio of PVA to fibroin. Though the preparative procedure of phase separation is relatively simple, safe and non-toxic, the blending solution of fibroin and PVA needs to be subjected to membrane forming, dissolving, washing and the like, and thus the process is relatively complicated.
[0091] The method according to the present invention is different from current phase separation method in principles, operation process, and drug applicability. The differences in principle mainly lie in the following: in phase separation, PVA solution and fibroin solution are mixed, and fibroin molecules are thoroughly dispersed in PVA solution by ultrasounding or stirring, then the dispersed solution is placed in an oven for drying and forming membrane and, after dissolving and washing, particles were obtained. On the other hand, the present invention uses the emulsion polymerization method, and fibroin solution is directly added in polyethylene glycol solution. With aid of its hydrophilicity, polyethylene glycol can rapidly absorb water molecules on surface of fibroin, and promote the hydrophobic regions of fibroin to approach each other, inducing aggregation among molecules to form self-assembled microspheres. The differences in operation process lies in the following: the phase separation method requires that the blending solution be placed in an oven at 60° C. for drying and forming membrane, followed by dissolving and washing, while the present invention only needs incubation at room temperature without forming a membrane. The differences in drug applicability lies in the following: fibroin particles prepared by phase separation have a low drug-loading ratio for hydrophobic neutral drugs, while polyethylene glycol used in the method of the present invention is an amphiphilic medicinal adjuvant, and hydrophobic neutral drugs can be dissolved in polyethylene glycol solution, then mixed and incubated with fibroin to form microspheres, thus the drug-loading ratio of fibroin particles is higher.
[0092] As far as controlled release of drugs, the drug-loaded fibrion nano/microparticles according to the present invention realize quantitive and zero-order or first-order release of drugs, and thus keep the blood level constant. By selecting small molecular drugs such as curcumin and adriamycin hydrochloride, large molecular drugs such as TMR-glucan, polypeptide and protein drugs such as octreotide and TMR-bovine serum albumin and the like and loading them on fibroin nano/microparticles by physical embedding, the present invention provides possibilities for clinical application of fibroin nano/microparticles as drug carrier in later stage.
EXAMPLE 1
1-1. Preparation of Fibroin Solution
[0093] 25 g of degummed, boiled-off silk was weighed and soaked in 9.3 M solution of LiBr (100 ml), and then placed in an oven at 60° C. for 4 h. The resulting mixture was stirred at one hour intervals till thorough dissolution, to obtain 20% (W/V) fibroin solution. The fibroin solution was poured into a Slide-a-lyzer dialysis bag with a molecular cut off of 3500, and the bag was transferred to a dialysis tank charged with deionized water and kept for 3 days. During dialysis, the deionized water was exchanged 10 times. After completion of dialysis, the fibroin solution was centrifuged under 9000 rpm/min at 4° C. for 20 min, to remove insoluble impurities. To determine the concentration of fibroin solution, a certain amount of fibroin solution was taken out and weighed, and then dried in an oven at 60° C. to dryness and weighed. The ratio of the two weights (fibroin solution and dried material) represents the concentration (mass percent, wt %). In general, the concentration of obtained fibroin solution was about 7 wt %. For the concentration of fibroin solution, the prepared fibroin solution (100 ml) was transferred into the same type of Slide-a-lyzer dialysis bag, and the bag was placed in a 15 wt % solution of polyethylene glycol having a molecular weight of 20000 (800 ml) for 24 h successive dialysis, to obtain about 30 wt % concentrated solution of fibroin, which was stored in a fridge at 4° C.
1.2. Preparation of Polyethylene Glycol-Fibroin Blending Solution
[0094] The 30 wt % fibroin solution prepared in 1-1 was diluted to 1%-5 wt %;
[0095] Polyethylene glycols having different molecular weight were prepared into solutions at different concentrations, and the concentration of polyethylene glycol having a molecular weight of less than 1000 was 40-100 wt %; the concentration of polyethylene glycol having a molecular weight of 1000-6000 was 10-60 wt %; the concentration of polyethylene glycol having a molecular weight of 10000 was 10-50 wt %; and the concentration of polyethylene glycol having a molecular weight of 20000 was 10-40 wt %;
[0096] Equal volume of 1-5 wt % fibroin solution was respectively taken out and added to same volume of polyethylene glycol solutions having different molecular weights and concentrations, and then gently agitated up and down with a pipette to mix the blending solution uniformly;
[0097] The polyethylene glycol-fibroin blending solution was incubated at room temperature for 30 min;
[0098] After incubation, the state of blending solution was observed and recorded.
1-3. Preparation of Polyethylene Glycol-Fibroin Blending Solution
[0099] The 30 wt % fibroin solution prepared in 1-1 was diluted to 6%-30 wt %;
[0100] Polyethylene glycols having different molecular weight were prepared into solutions at different concentrations, and the concentration of polyethylene glycol having a molecular weight of 2000-6000 was 10-60 wt %; the concentration of polyethylene glycol having a molecular weight of 10000 was 10-50 wt %; and the concentration of polyethylene glycol having a molecular weight of 20000 was 10-40 wt %;
[0101] Equal volume of 6-30 wt % fibroin solution was respectively taken out and added to same volume of polyethylene glycol solutions having different molecular weights and concentrations, and then gently agitated up and down with a pipette to mix the blending solution uniformly;
[0102] The polyethylene glycol-fibroin blending solution was incubated at room temperature for 30 min;
[0103] After incubation, the state of blending solution was observed and recorded.
[0104] Experimental results of 1-1 and 1-2 were summarized in Table 1. From the recorded values, it can be seen that polyethylene glycols with high molecular weight readily mixed with the fibroin solution to form particles; the molecular weight and the concentration of polyethylene glycol as well as the concentration of fibroin are direct factors affecting the particle formation.
TABLE-US-00001 TABLE 1 State of the blending solution 30 minutes after the mixing of fibroin solution and polyethylene glycol solution PEG molecular PEG Fibroin Change of solution state weight concentration concentration after blending for 30 (Da) (wt %) (wt %) minutes 200 100 1-5 1 wt % solution, 2-5 wt % gel-forming 50 1-5 1-4 wt % solution, 45 1-5 5 wt % gel-forming 40 1-5 300 100 1-5 1-5 wt % gel-forming 55 1-5 1-2 wt % solution, 3-5 wt % gel-forming 50 1-5 1-3 wt % solution, 45 1-5 4-5 wt % gel-forming 400 100 1-5 1-5 wt % gel-forming 55 1-5 1 wt % solution, 50 1-5 2-5 wt % gel-forming 45 1-5 600 100 1-5 1-5 wt % gel-forming 60 1-5 1-2 wt % solution, 3-5 wt % gel-forming 55 1-5 1-3 wt % solution, 4-5 wt % gel-forming 50 1-5 1-5 wt % solution 1000 60 1-5 1 wt % solution, 2-5 wt % gel-forming 55 1-5 1-2 wt % solution, 3-5 wt % gel-forming 10 1-5 1-5 wt % solution 1500 60 1-5 1 wt % solution, 2-5 wt % gel-forming 55 1-5 1-2 wt % solution, 3-5 wt % gel-forming 10 1-5 1-3 wt % solution, 4-5 wt % gel-forming 2000 60 1-30 1 wt % solution, 2-30 wt % gel-forming 50 1-30 1-2 wt % solution, 3-30 wt % gel-forming 40 1-30 1-2 wt % solution, 3-30 wt % particle-forming 10 1-30 1-30 wt % solution 4000 60 1-30 1-3 wt % solution, 4-30 wt % particle-forming 40 1-30 1-4 wt % solution, 5-30 wt % particle-forming 10 1-30 1-30 wt % solution 6000 60 1-30 1-3 wt % solution, 4-30 wt % particle-forming 40 1-30 1-3 wt % solution, 4-30 wt % particle-forming 10 1-30 1-30 wt % solution 10000 50 1-30 1-2 wt % solution, 3-30 wt % particle-forming 25 1-30 1-3 wt % solution, 4-30 wt % particle-forming 10 1-30 1-4 wt % solution, 5-30 wt % particle-forming 20000 40 1-30 1-2 wt % solution, 3-30 wt % particle-forming 20 1-30 1-3 wt % solution, 4-30 wt % particle-forming 10 1-30 1-30 wt % solution
1-4. Preparation of Fibroin Nano/Microspheres
[0105] The following polyethylene glycol solutions were prepared separately: 40 wt % solution of polyethylene glycol having a molecular weight of 2000; 40 wt % solution of polyethylene glycol having a molecular weight of 4000; 40 wt % solution of polyethylene glycol having a molecular weight of 6000; 40 wt % solution of polyethylene glycol having a molecular weight of 10000; and 40 wt % solution of polyethylene glycol having a molecular weight of 20000.
[0106] The 30 wt % fibroin solution prepared in 1-1 was diluted to 8 wt %, and the diluted solution was added to equal volume of polyethylene glycol solutions with concentration and molecular weight described above;
[0107] The polyethylene glycol-fibroin blending solution was incubated at room temperature for 12 h;
[0108] After incubation, the centrifuge tubes were placed in a centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0109] Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0110] Suspension of fibroin nano/microspheres was prepared using deionized water and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0111] It can be seen from
1-5. Determination of Particle Size of Fibroin Nano/Microspheres
[0112] Suspensions of fibroin nano/microspheres prepared in 1-4 were respectively taken out and transferred to disposable cuvettes;
[0113] The particle size of fibroin nano/microspheres in each group was measured using laser particle size analyzer in triplicate, and the average value was calculated.
[0114] As can be seen from
1-6. Preparation of Fibroin Nano/Microspheres
[0115] 1. 30% solution of polyethylene glycol having a molecular weight of 4000 was prepared (indicating the molecular weight of polyethylene glycol is 4000, and the concentration is 30 wt %);
[0116] 2. Fibroin solution at a concentration of 5% was prepared according to the method of Example 1.
[0117] 3. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 2:1, and the mixture was incubated for 24 h at a temperature of 25° C., 45° C. and 60° C., respectively;
[0118] 4. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0119] 5. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0120] 6. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0121] With reference to
EXAMPLE 2
[0122] 1. The polyethylene glycol solutions with different molecular weight and same concentration in examples 1-4 are selected;
[0123] 2. The viscosity of above polyethylene glycol solutions was respectively measured using rheometer;
[0124] 3. Fibroin solution at a concentration of 8% was prepared according to the method of Example 1;
[0125] 4. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 1:1, the mixture was gently agitated to blend uniformly, and then incubated for 12 h at room temperature;
[0126] 5. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0127] 6. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0128] 7. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0129] With reference to
EXAMPLE 3
[0130] 1. 50% solution of polyethylene glycol having a molecular weight of 10000 was prepared (indicating the molecular weight of polyethylene glycol is 10000, and the concentration is 50 wt %) and then diluted to 20% and 30%, respectively;
[0131] 2. The viscosity of above polyethylene glycol solutions at different concentrations was respectively measured using rheometer;
[0132] 3. Fibroin solution at a concentration of 8% was prepared according to the method of Example 1;
[0133] 4. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 1:1, and the mixture was incubated for 24 h at room temperature;
[0134] 5. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0135] 6. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0136] 7. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0137] With reference to
[0138] With reference to
EXAMPLE 4
[0139] 1. 50 wt % solution of polyethylene glycol having a molecular weight of 10000 was prepared;
[0140] 2. Fibroin solutions at concentrations of 1%, 2%, 5%, 8%, 12%, and 20 wt % were prepared respectively according to the method of Example 1,
[0141] 3. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 1:1, and the mixture was incubated for 2 h at room temperature;
[0142] 4. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0143] 5. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0144] 6. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0145] With reference to
[0146] With reference to
EXAMPLE 5
[0147] 1. 50 wt % solution of polyethylene glycol having a molecular weight of 10000 was prepared;
[0148] 2. Fibroin solution at concentration of 8 wt % was prepared according to the method of Example 1;
[0149] 3. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 10/10/1 (SF/polyethylene glycol/methanol solution), and the mixture was incubated for 2 h at room temperature;
[0150] 4. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0151] 5. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0152] 6. Suspension of fibroin nano/microspheres was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0153] With reference to
EXAMPLE 6
[0154] 1. 60% solution of polyethylene glycol having a molecular weight of 4000 was prepared (indicating the molecular weight of polyethylene glycol is 4000, and the concentration is 60 wt %);
[0155] 2. Fibroin solutions at concentration of 1% and 5% were prepared respectively;
[0156] 3. The pH value of above fibroin solutions was adjusted to 3.6 and 7.0 respectively;
[0157] 4. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 1/1, and the mixture was incubated for 24 h at room temperature;
[0158] 5. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0159] 6. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0160] 7. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0161] With reference to
EXAMPLE 7
[0162] 1. 50% solution of polyethylene glycol having a molecular weight of 10000 was prepared (indicating the molecular weight of polyethylene glycol is 10000, and the concentration is 50 wt %);
[0163] 2. Fibroin solutions at concentration of 0.2%, 1%, 5%, 10% and 15% were prepared, respectively;
[0164] 3. The pH value of above fibroin solutions was adjusted to 3.6, 7.0 and 10.0, respectively;
[0165] 4. Fibroin solution was mixed with the solution of polyethylene glycol at a volume ratio of 1:1, and the mixture was incubated for 24 h at room temperature;
[0166] 5. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0167] 6. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0168] 7. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0169] With reference to
[0170] With reference to
EXAMPLE 8
[0171] 1. 20% solution of polyethylene glycol having a molecular weight of 10000 was prepared (indicating the molecular weight of polyethylene glycol is 10000, and the concentration is 20 wt %);
[0172] 2. Solutions of sodium chloride, potassium chloride and magnesium chloride at a concentration of 1 mol/L were prepared, respectively;
[0173] 3. Lyophilized powder was dissolved in deionized water, and the final concentration was 30%;
[0174] 4. Equal volume of above salt ion solutions was added so that the final concentration of fibroin solution is 15%, and the final concentration of salt ions is 0.5 M;
[0175] 5. Fibroin/salt ions solution was mixed with the solution of polyethylene glycol at a volume ratio of 1:1, and the mixture was shaken gently to mix uniformly;
[0176] 6. Absolute ethanol solution (V.sub.above blending solution/V.sub.ethanol=1/4) was added quickly, and after uniform mixing, the mixture was rapidly added to a culture dish and then transferred to an oven at 60° C.;
[0177] 7. After drying, the culture dish was taken out, to which deionized water was added to make suspension;
[0178] 8. The suspension was sonicated for 2 min using ultrasonic cell disruptor, with 30% amplitude of vibration;
[0179] 9. Centrifugating: room temperature, 20 min, 12000 rpm;
[0180] 10. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0181] 11. Suspension of fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0182] With reference to
[0183] With reference to
[0184] With reference to
[0185] With reference to
[0186] With reference to
[0187] With reference to
EXAMPLE 9
[0188] 1. Weighing 0.1 mg of dry fibroin nano/microparticles obtained after freeze-drying in examples 4 and 5, respectively;
[0189] 2. Taking small amount of potassium bromide powder and mixing with above fibroin nano/microparticles uniformly, grinding evenly with a pestle, and then tabletting with a tablet press;
[0190] 3. Measuring with Fourier infrared spectrometer under the following conditions: surface total reflection, wave number range: 400-4000 cm.sup.−1, and thereby, the infrared structure of each sample was tested;
[0191] 4. Taking fibroin nano/microparticles prepared from 8% fibroin solution (with or without addition of methanol) and pure lyophilized fibroin powder (without adding polyethylene glycol and methanol, as control), and the secondary structure content of each sample was subjected to simulation analysis using Peakfit software.
[0192] With reference to
[0193] With reference to
EXAMPLE 10
[0194] 1. Weighing 0.1 mg of dry fibroin nano/microparticles obtained after freeze-drying in examples 1-6, respectively;
[0195] 2. Taking small amount of potassium bromide powder and mixing with above fibroin nano/microparticles uniformly, grinding evenly with a pestle, and then tabletting with a tablet press;
[0196] 3. Measuring with Fourier infrared spectrometer under the following conditions: surface total reflection, wave number range: 400-4000 cm.sup.−1, and thereby, the infrared structure of each sample was tested;
[0197] 4. Choosing IR spectra of above fibroin nano/microparticles, and the secondary structure content of each sample was subjected to simulation analysis using Peakfit software.
[0198] With reference to
[0199] With reference to
EXAMPLE 11
[0200] 1. Weighing 0.1 mg of dry fibroin nano/microparticles obtained after freeze-drying in example 8;
[0201] 2. Taking small amount of potassium bromide powder and mixing with above fibroin nano/microparticles uniformly, grinding evenly with a pestle, and then tabletting with a tablet press;
[0202] 3. Measuring with Fourier infrared spectrometer under the following conditions: surface total reflection, wave number range: 400-4000 cm.sup.−1, and thereby, the infrared structure of each sample was tested;
[0203] 4. Choosing IR spectra of above fibroin nano/microparticles, and the secondary structure content of each sample was subjected to simulation analysis using Peakfit software.
[0204] With reference to
[0205] With reference to
EXAMPLE 12
[0206] 1. Weighing 0.1 mg of dry fibroin nano/microparticles obtained after freeze-drying in example 9;
[0207] 2. Taking small amount of potassium bromide powder and mixing with above fibroin nano/microparticles uniformly, grinding evenly with a pestle, and then tabletting with a tablet press;
[0208] 3. Measuring with Fourier infrared spectrometer under the following conditions: surface total reflection, wave number range: 400-4000 cm.sup.−1, and thereby, the infrared structure of each sample was tested;
[0209] With reference to
[0210] With reference to
[0211] With reference to
EXAMPLE 13
Preparation of Curcumin-Fibroin Nano/Microparticles
[0212] 1. Weighing 0.5 mg and 0.9 mg of curcumin, respectively;
[0213] 2. Taking 50 wt % solution of polyethylene glycol having a molecular weight of 10000 (1 ml each), to respectively dissolve curcumin;
[0214] 3. Preparing 5 wt % and 9 wt % fibroin solutions, 1 ml each;
[0215] 4. Adding the fibroin solutions to above curcumin-polyethylene glycol solutions respectively, and gently agitating up and down with pipette, to mix the blending solution uniformly;
[0216] 5. The blending solution of curcumin-polyethylene glycol-fibroin was left at room temperature and incubated for 2 h;
[0217] 6. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0218] 7. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0219] 8. Suspension of curcumin-fibroin nano/microparticles was prepared and stored after vacuum freeze-drying;
[0220] 9. Respectively, 1 mg of curcumin-fibroin nano/microparticles prepared with 5 wt % and 9 wt % fibroin solutions was weighed and added to 1 ml methanol solution, three samples per group;
[0221] 10. Centrifuge tubes were vortexed for 10 min, and then placed in a rotator to rotate for 2 h at 60° C.;
[0222] 11. Centrifuge tubes were taken out and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0223] 12. The supernatant was collected, and the absorbance of curcumin was measured at 425 nm using a microplate reader, then the content of curcumin in fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for curcumin, thereby the drug-loading ratio was calculated;
[0224] 13. The drug-loading ratio of curcumin-fibroin nano/microparticles prepared with 5% fibroin solution was 0.27%±0.07%; while the drug-loading ratio of curcumin-fibroin nano/microparticles prepared with 9% fibroin solution was 0.51%±0.15%.
[0225] With reference to
EXAMPLE 14
Preparation of TMR-Dextran-Fibroin Nano/Microparticles
[0226] 1. TMR-dextran was dissolved in water to prepare 1.0 mg/ml and 1.8 mg/ml solutions respectively;
[0227] 2. 10 wt % and 18 wt % of fibroin solutions were prepared respectively;
[0228] 3. The above TMR-dextran solution was mixed equal volume of fibroin solution, thereby the weight ratio of fibroin and TMR-dextran was 100/1;
[0229] 4. 50 wt % solution of polyethylene glycol having a molecular weight of 10000 was prepared;
[0230] 5. The blending solution of fibroin and TMR-dextran was mixed with equal volume of the polyethylene glycol solution, and gently agitating up and down with pipette, to mix the blending solution uniformly;
[0231] 6. The blending solution of TMR-dextran-polyethylene glycol-fibroin was left at room temperature and incubated for 2 h;
[0232] 7. After completion of incubation, the centrifuge tubes were placed in a centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0233] 8. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0234] 9. Suspension of TMR-dextran-fibroin nano/microparticles was prepared and stored after vacuum freeze-drying;
[0235] 10. Respectively, 1 mg of TMR-dextran-fibroin nano/microparticles prepared with 5 wt % and 9 wt % fibroin solutions was weighed and added to 1 ml lithium bromide solution, three samples per group;
[0236] 11. Centrifuge tubes were vortexed for 10 min, and then placed in a rotator to rotate for 2 h at 60° C., avoiding exposure to light;
[0237] 12. Centrifuge tubes were taken out and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0238] 13. The supernatant was collected, and the fluorescence value of TMR-dextran was measured at excitation wavelength of 550 nm and emission wavelength of 590 nm using ELISA instrument, then the content of TMR-dextran in fibroin nano/microparticles was calculated by comparing with fluorescence value-concentration standard curve for TMR-dextran, thereby the drug-loading ratio was calculated;
[0239] 14. The drug-loading ratio of TMR-dextran-fibroin nano/microparticles prepared with 5% fibroin solutions was 0.13%; while the drug-loading ratio of TMR-dextran-fibroin nano/microparticles prepared with 9% fibroin solutions was 0.26%.
[0240] With reference to
EXAMPLE 15
Preparation of CdTe-Fibroin Nano/Microparticles
[0241] 1. Solution of fibroin and CdTe quantum dots with a concentration ratio of 0.017 nmol CdTe/1 mg SF was prepared using deionized water, wherein the final concentration of fibroin was 5 wt %, 8 wt %, and 20 wt %;
[0242] 2. 50 wt % solution of polyethylene glycol having a molecular weight of 10000 was prepared;
[0243] 3. The blending solution of fibroin and CdTe was mixed with equal volume of the polyethylene glycol solution, and gently agitating up and down with pipette, to mix the blending solution uniformly;
[0244] 4. The blending solution of CdTe-fibroin-polyethylene glycol was left at room temperature and incubated for 2 h;
[0245] 5. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0246] 6. Deionized water was added for washing, the supernatant was removed and kept for testing, and then the centrifugation and washing operations were repeated three times;
[0247] 7. The supernatant was collected, and the fluorescence value of CdTe quantum dots was measured at excitation wavelength of 380 nm and emission wavelength of 596 nm using a microplate reader, then the content of CdTe in supernatant was calculated by comparing with fluorescence value-concentration standard curve for CdTe, thereby the encapsulation ratio was calculated;
[0248] 8. Suspension of CdTe-fibroin nano/microparticles was prepared and stored after vacuum freeze-drying;
[0249] 9. The encapsulation ratio of CdTe-fibroin nano/microparticles prepared with 5% fibroin solution was 98.47%±0.17%; the encapsulation ratio of CdTe-fibroin nano/microparticles prepared with 8% fibroin solution was 98.89%±0.23%; and the encapsulation ratio of CdTe-fibroin nano/microparticles prepared with 20% fibroin solution was 96.82%±0.27%
[0250] With reference to
EXAMPLE 16
Preparation of Curcumin-Fibroin Nano/Microparticles
[0251] 1. Weighing 1 mg of curcumin;
[0252] 2. Taking 1 ml of 50 wt % solution of polyethylene glycol having a molecular weight of 10000 to dissolve curcumin;
[0253] 3. Preparing solution of fibroin at a concentration of 5 wt % and adjusting its pH to 3.6 and 7.0;
[0254] 4. Adding the fibroin solutions to above curcumin-polyethylene glycol solution respectively and gently agitating up and down with pipette, to mix the blending solution uniformly;
[0255] 5. The blending solution of curcumin-polyethylene glycol-fibroin was left at room temperature and incubated for 24 h;
[0256] 6. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0257] 7. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0258] 8. Suspension of curcumin-fibroin nano/microparticles was prepared and stored after vacuum freeze-drying;
[0259] 9. Weighing 1 mg of curcumin-fibroin nano/microparticles prepared with 5% fibroin solutions at pH 3.6 and 7.0 respectively and adding to 1 ml methanol solution, three samples per group;
[0260] 10. Centrifuge tubes were vortexed for 10 min, and then placed in a rotator to rotate for 2 h;
[0261] 11. Centrifuge tubes were taken out and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0262] 12. The supernatant was collected, and the absorbance of curcumin was measured at 425 nm using a microplate reader, then the content of curcumin in fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for curcumin, thereby the drug-loading ratio was calculated;
[0263] 13. The drug-loading ratio of curcumin-fibroin nano/microparticles prepared with 5% fibroin solution at pH 3.6 was 1.18%±0.08%; while the drug-loading ratio of curcumin-fibroin nano/microparticles prepared with 5% fibroin solution at pH 7.0 was 0.64%±0.06%.
[0264] With reference to
[0265] With reference to
EXAMPLE 17
Preparation of Doxorubicin Hydrochloride-Fibroin Nano/Microparticles
[0266] 1. Weighing 1 mg of doxorubicin hydrochloride;
[0267] 2. Preparing solutions of fibroin at a concentration of 5% and pH values of 3.6 and 7.0;
[0268] 3. Dissolving the doxorubicin hydrochloride particles in above fibroin solutions;
[0269] 4. Preparing 50 wt % solution of polyethylene glycol having a molecular weight of 10000;
[0270] 5. Adding fibroin solutions containing doxorubicin hydrochloride to the polyethylene glycol solution and gently agitating up and down with pipette, to mix the blending solution uniformly;
[0271] 6. The blending solution of doxorubicin hydrochloride-fibroin-polyethylene glycol was left at room temperature and incubated for 24 h;
[0272] 7. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0273] 8. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0274] 9. Suspension of doxorubicin hydrochloride-fibroin nano/microparticles was prepared and stored after vacuum freeze-drying;
[0275] 10. Weighing 1 mg of doxorubicin hydrochloride-fibroin nano/microparticles prepared with 5% fibroin solutions at pH 3.6 and 7.0 respectively and adding to 1 ml lithium bromide solution, three samples per group;
[0276] 11. Centrifuge tubes were vortexed for 10 min, and then placed in a rotator to rotate for 2 h at 60° C.;
[0277] 12. Centrifuge tubes were taken out and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0278] 13. The supernatant was collected, and the absorbance of doxorubicin hydrochloride was measured at 600 nm using a microplate reader, then the content of doxorubicin hydrochloride in fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for doxorubicin hydrochloride, thereby the drug-loading ratio was calculated;
[0279] 14. The drug-loading ratio of doxorubicin hydrochloride-fibroin nano/microparticles prepared with 5% fibroin solution at pH 3.6 was 0.80%±0.06%; while the drug-loading ratio of doxorubicin hydrochloride-fibroin nano/microparticles prepared with 5% fibroin solution at pH 7.0 was 1.90%±0.05%.
[0280] With reference to
[0281] With reference to
EXAMPLE 18
[0282] 1. Weighing 10 mg of curcumin-fibroin nano/microparticles prepared in example 13 and adding to centrifuge tubes;
[0283] 2. Phosphate buffer with a molar concentration of 0.01 mol/L (PBS, pH=7.4) was prepared, and then 5% Tween-80 and 3% methanol were added to prepare releasing solution;
[0284] 3. Adding 1 ml of above releasing solution to each sample respectively;
[0285] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0286] 5. Centrifuging at room temperature, 12000 rpm for 10 min;
[0287] 6. The supernatant was collected, and the absorbance of curcumin was measured at 425 nm using a microplate reader, then the release amount of curcumin from fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for curcumin, thereby the cumulative release ratio was calculated.
[0288] With reference to
EXAMPLE 19
[0289] 1. Weighing 10 mg of TMR-dextran-fibroin nano/microparticles prepared in example 14 and adding to centrifuge tubes;
[0290] 2. Phosphate buffer with a molar concentration of 0.01 mol/L (PBS, pH=7.4) was prepared;
[0291] 3. Adding 1 ml of PBS releasing solution to each sample respectively;
[0292] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0293] 5. Centrifuging at room temperature, 12000 rpm for 10 min;
[0294] 6. The supernatant was collected, and the fluorescence value of TMR-dextran was measured at excitation wavelength of 555 nm and emission wavelength of 590 nm using a microplate reader, then the release amount of TMR-dextran from fibroin nano/microparticles was calculated by comparing with fluorescence value-concentration standard curve for TMR-dextran, thereby the cumulative release ratio was calculated.
[0295] With reference to
EXAMPLE 20
[0296] 1. Weighing 10 mg of CdTe-fibroin nano/microparticles prepared in example 15 and adding to centrifuge tubes;
[0297] 2. Adding 1 ml of deionized water to each sample respectively;
[0298] 3. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0299] 4. Centrifuging at room temperature, 12000 rpm for 10 min;
[0300] 5. The supernatant was collected, and the fluorescence value of CdTe quantum dots was measured at excitation wavelength of 380 nm and emission wavelength of 596 nm using a microplate reader, then the release amount of CdTe quantum dots from fibroin nano/microparticles was calculated by comparing with fluorescence value-concentration standard curve for CdTe quantum dots, thereby the cumulative release ratio was calculated.
[0301] With reference to
EXAMPLE 21
[0302] 1. Weighing 10 mg of curcumin-fibroin nano/microparticles prepared in example 16 and adding to centrifuge tubes;
[0303] 2. Phosphate buffer with a molar concentration of 0.01mol/L (PBS, pH=7.4) was prepared, and then 5% Tween-80 and 3% methanol were added to prepare releasing solution;
[0304] 3. Adding 1 ml of above releasing solution to each sample respectively;
[0305] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0306] 5. Centrifuging at room temperature, 12000 rpm for 10 min;
[0307] 6. The supernatant was collected, and the absorbance of curcumin was measured at 425 nm using a microplate reader, then the release amount of curcumin from fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for curcumin, thereby the cumulative release ratio was calculated.
[0308] With reference to
EXAMPLE 22
[0309] 1. Weighing 10 mg of doxorubicin hydrochloride-fibroin nano/microparticles prepared in example 17 and adding to centrifuge tubes;
[0310] 2. Phosphate buffer with a molar concentration of 0.01 mol/L (PBS, pH=4.0) was prepared;
[0311] 3. Adding 1 ml of PBS releasing solution to each sample respectively;
[0312] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0313] 5. Centrifuging at room temperature, 12000 rpm for 10 min;
[0314] 6. The supernatant was collected, and the absorbance of doxorubicin hydrochloride was measured at 480 nm using a microplate reader, then the release amount of doxorubicin hydrochloride from fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for doxorubicin hydrochloride, thereby the cumulative release ratio was calculated.
[0315] With reference to
EXAMPLE 23
[0316] 1. Weighing 10 mg of doxorubicin hydrochloride-fibroin nano/microparticles prepared in example 17 and adding to centrifuge tubes;
[0317] 2. Phosphate buffer with a molar concentration of 0.01 mol/L (PBS, pH=7.4) was prepared;
[0318] 3. Adding 1 ml of PBS releasing solution to each sample respectively;
[0319] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0320] 5. Centrifugating at room temperature, 12000 rpm for 10 min;
[0321] 6. The supernatant was collected, and the absorbance of doxorubicin hydrochloride was measured at 480 nm using a microplate reader, then the release amount of doxorubicin hydrochloride from fibroin nano/microparticles was calculated by comparing with absorbance-concentration standard curve for doxorubicin hydrochloride, thereby the cumulative release ratio was calculated.
[0322] With reference to
EXAMPLE 24
Preparation of Octreotide-Fibroin Nano/Microparticles
[0323] 1. Preparing 20 mg/ml octreotide solution with deionized water;
[0324] 2. Dissolving lyophilized fibroin powder in deionized water, and the concentration was 100 mg/ml;
[0325] 3. Blending the above solutions (2 ml each), thereby the weight ratio of fibroin to octreotide was 5/1;
[0326] 4. 4 ml of 50 wt % solution of polyethylene glycol having a molecular weight of 10000 was taken out;
[0327] 5. The above three solutions were mixed together and incubated at room temperature for 24 h;
[0328] 6. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0329] 7. The supernatant was reserved, and the centrifugation and washing operations were repeated three times, then was suspended and freeze-dried;
[0330] See
EXAMPLE 25
Preparation of TMR-Bovine Serum Albumin-Fibroin Nano/Microparticles
[0331] 1. TMR-bovine serum albumin solution (1 ml) at a concentration of 0.5 mg/ml was prepared with deionized water;
[0332] 2. Lyophilized fibroin powder was dissolved with deionized water, and the concentration was 60 mg/ml, the volume was 5 ml;
[0333] 3. The above solutions were mixed, wherein the weight ratio of fibroin to TMR-bovine serum albumin was 600/1;
[0334] 4. 6 ml of 50% solution of polyethylene glycol having a molecular weight of 10000 was taken out;
[0335] 5. The above three solutions were mixed together and incubated at room temperature for 24 h;
[0336] 6. After completion of incubation, the centrifuge tubes were placed in centrifugal machine and centrifuged at room temperature, 12000 rpm/min for 10 min;
[0337] 7. The supernatant was reserved, and the centrifugation and washing operations were repeated three times, then was suspended and freeze-dried;
[0338] 8. The content of TMR-bovine serum albumin in supernatant was measured, the fluorescence value of TMR-bovine serum albumin was measured at excitation wavelength of 550 nm and emission wavelength of 590 nm using a microplate reader and the fluorescence-concentration standard curve was plotted, then the fluorescence of test sample was measured and the content of TMR-bovine serum albumin in supernatant was calculated, thereby the encapsulation ratio of TMR-bovine serum albumin was calculated, which was 40.4±4.2%.
[0339] See
[0340] See
EXAMPLE 26
[0341] 1. Weighing 5 mg of TMR-bovine serum albumin-fibroin nano/microparticles prepared in example 25;
[0342] 2. Phosphate buffer with a molar concentration of 0.01 mol/L (PBS, pH=7.4) was prepared;
[0343] 3. Adding 1 ml of PBS releasing solution to each sample respectively;
[0344] 4. The mixture was placed in an oven at 37° C. and allowed to release drugs under shaking, and was taken out at indicated time;
[0345] 5. Centrifuging at room temperature, 12000 rpm for 10 min;
[0346] 6. The supernatant was collected, and the fluorescence value of TMR-bovine serum albumin was measured at excitation wavelength of 555 nm and emission wavelength of 590 nm using a microplate reader, then the release amount of TMR-bovine serum albumin from fibroin nano/microparticles was calculated by comparing with fluorescence value-concentration standard curve for TMR-bovine serum albumin, thereby the cumulative release ratio was calculated.
[0347] With reference to
EXAMPLE 27
[0348] 1. 20% solution of polyethylene glycol having a molecular weight of 10000 was prepared (indicating the molecular weight of polyethylene glycol is 10000, and the concentration is 20 wt %)
[0349] 2. Solutions of sodium chloride, potassium chloride and magnesium chloride at a concentration of 0.2 mol/L were prepared, respectively;
[0350] 3. Lyophilized powder was dissolved in deionized water, and the final concentration was 30%;
[0351] 4. 24 mg of doxorubicin hydrochloride was weighed and dissolved with deionized water (4 ml) at room temperature, to obtain doxorubicin hydrochloride solution at 6 mg/ml;
[0352] 5. To the above doxorubicin hydrochloride solution of 6 mg/ml, equal volume of above salt ion solutions was added to obtain an doxorubicin hydrochloride solution at a final concentration of 3 mg/ml;
[0353] 6. The above fibroin solution was diluted with equal volume of above doxorubicin hydrochloride solution dissolved with above salt ions solutions, to obtain a 15% fibroin solution wherein the concentration of doxorubicin was 1.5 mg/ml, the concentration of salt ions was 0.1 mol/L (the weight ratio of doxorubicin hydrochloride to fibroin was 1/100);
[0354] 7. The solution of doxorubicin-fibroin, treated with salt ions, was mixed with the solution of polyethylene glycol at a volume ratio of 1/1, and then gently shaken to mix uniformly;
[0355] 8. Absolute ethanol solution (V.sub.above blending solution/V.sub.ethanol=1/4) was added quickly, and after uniform mixing, the mixture was rapidly added to a culture dish and then transferred to an oven at 60° C.;
[0356] 9. After drying, the culture dish was taken out, to which deionized water was added to make suspension;
[0357] 10. The suspension was sonicated for 2 min using ultrasonic cell disruptor, with 30% amplitude of vibration;
[0358] 11. Centrifuging: room temperature 20 min, 12000 rpm;
[0359] 12. Deionized water was added for washing, the supernatant was removed, and the centrifugation and washing operations were repeated three times;
[0360] 13. Suspension of doxorubicin hydrochloride-fibroin nano/microparticles was prepared and stored in a fridge at 4° C., or stored after vacuum freeze-drying.
[0361] With reference to
[0362] With reference to
[0363] With reference to
[0364] With reference to
[0365] With reference to
[0366] With reference to
[0367] The above are only preferred embodiments of the present invention, and the invention is not limited to these. It should be pointed out that ordinary skilled persons in the art can make various improvements and variations without departing from the technical principle of the invention, and such improvements and variations should also be regarded as under the protection of the invention.