NANOPARTICLES AND METHOD FOR MANUFACTURING THE SAME

Abstract

Disclosed herein are nanoparticles and method for manufacturing the same. The nanoparticle is essentially composed of albumin and polyethylene glycol, wherein the albumin is covalently crosslinked with the polyethylene glycol.

Claims

1. A nanoparticle, essentially composed of albumin and polyethylene glycol, wherein the albumin is covalently crosslinked with the polyethylene glycol.

2. The nanoparticle according to claim 1, further comprising a radioactive material shot to a surface of the nanoparticle.

3. The nanoparticle according to claim 2, wherein the radioactive material is rhenium-188 or technetium-99 m.

4. The nanoparticle according to claim 1, wherein the albumin is human serum albumin.

5. The nanoparticle according to claim 1, wherein the polyethylene glycol has a maleimide functional group.

6. A method for manufacturing a nanoparticle, comprising: a) dissolving human serum albumin and polyethylene glycol in a PBS solution, to obtain a mixture; b) dissolving the mixture to a saline solution, and adding 0.05-0.2 N of sodium hydroxide; c) adding a methanol/ethanol mixture for reaction; d) after precipitation of the albumin, adding a glutaraldehyde water solution for reaction; and e) obtaining the nanoparticle through ultracentrifugation.

7. The method according to claim 6, wherein centrifugation is carried out after step a) reacts for 20 hours.

8. The method according to claim 6, wherein step b) is completed to filter to obtain a filtering medium, and then step c) is performed.

9. The method according to claim 6, wherein centrifugation is carried out after step d) reacts for 12 hours.

10. The method according to claim 6, further comprising adding the nanoparticle to a trhenium-188 solution, filling the trhenium-188 solution with nitrogen and then carrying out a reaction, to obtain a nanoparticle that marks trhenium-188.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0024] To make the aforementioned and other objectives, features, advantages and examples of the present invention more comprehensible, the description of the drawings is as follows:

[0025] FIG. 1A shows results of mass spectrometry analysis on polyethylene glycol (PEG)-HSA (denoted by HSA/mal-PEG in the figure) of the present invention according to one embodiment of the present invention;

[0026] FIG. 1B shows results of mass spectrometry analysis on HSA according to one embodiment of the present invention;

[0027] FIG. 2 shows results of analysis on particle size of a nanoparticle of the present invention according to another embodiment of the present invention;

[0028] FIG. 3A shows results of analysis on particle size of a nanoparticle marking a radioactive material of the present invention according to another embodiment of the present invention; and

[0029] FIG. 3B shows results of analysis on surface potential of the nanoparticle marking a radioactive material of the present invention shown in FIG. 3A.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

[0030] To make statement of the disclosure more detailed and complete, illustrative literal statement is put forward hereinafter for implementation aspects and specific examples of the present invention; however, the implementation aspects and specific examples of the present invention are not merely limited thereto.

[0031] Unless otherwise specified, meanings of scientific and technical terms used in the specification are the same as those understood and commonly used by those of ordinary skill in the art. Moreover, the nouns used in the specification all cover singular and plural forms of the nouns, unless other indicated.

[0032] As stated in the specification, the word about usually means that an actual value is within 10%, 5%, 1% or 0.5% of a particular value or range. The word about herein represents that an actual value falls within an acceptable standard error of an average value, which depends on consideration of those of ordinary skill in the art. Except experiments, or unless otherwise explicitly specified, it should be understood that the range, number, value and percentage used herein are all modified by about. Therefore, unless otherwise specified, values or parameters disclosed in the specification and the appended claims are all approximate values, and may vary as required.

[0033] The present invention proposes a new nanoparticle, which can improve the problem of poor stability of the albumin nanoparticle. On the other hand, the present invention proposes a method for manufacturing a nanoparticle, so that the nanoparticle of the present invention can be obtained without using poisonous substances such as trichloromethane or dichloromethane during manufacturing the albumin nanoparticle, that is, the past tedious manufacturing process is simplified, to achieve the aim of enhancing the efficiency of manufacturing the albumin nanoparticle and reducing the risk that the nanoparticle is contaminated by highly toxic chemicals. In addition, the present invention further proposes a new method for manufacturing a nanoparticle marked with a radioactive material, and the method is different from the prior art in that it can be completed without a dispersing agent and a stabilizer.

[0034] The nanoparticle of the present invention, structurally, is essentially composed of albumin and polyethylene glycol, wherein the albumin is covalently crosslinked with the polyethylene glycol. In one embodiment, the nanoparticle of the present invention can serve as a carrier, to transfer a preparation to a target area. The preparation may be a therapeutic agent or a developing agent, wherein the therapeutic agent is a chemotherapy drug or a radiotherapy drug. In any optional embodiment, the chemotherapy drug may be a liposoluble chemotherapy drug or a non-liposoluble chemotherapy drug, and those of ordinary skill in the art can select a suitable effective chemotherapy drug according to actual use conditions, for example, Taxol, erlotinib, gefitinib and the like.

[0035] In one preferred embodiment, the nanoparticle of the present invention may be combined with the radiotherapy drug, to serve as a radioactive isotopic carrier. The advantages of the nanoparticle with a radioactive material of the present invention are as follows: (1) the nanoparticle has biodegradability and biocompatibility, which eliminates security concerns remaining in the body; and (2) the nanoparticle can carry out a marking reaction in high-temperature environments, which facilitates radioactive nuclide marking.

[0036] In any optional embodiment, the radiotherapy drug or radioactive material may be rhenium-188 or technetium-99 m.

[0037] In addition, the specific steps of the manufacturing method of the present invention are as follows:

[0038] a) dissolving human serum albumin and polyethylene glycol in a PBS solution, to obtain a mixture;

[0039] b) dissolving the mixture to a saline solution, and adding 0.05-0.2 N of sodium hydroxide;

[0040] c) adding a methanol/ethanol mixture for reaction;

[0041] d) after precipitation of the albumin, adding a glutaraldehyde water solution for reaction; and

[0042] e) obtaining the nanoparticle through ultracentrifugation.

[0043] In one embodiment of the present invention, a mal ratio of HSA to PFG in step a) is about 1:2 to 1:10, for example, about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10. In one preferred embodiment, the mal ratio is about 1:3 to 1:6; in one more preferred embodiment, the mal ratio is about 1:5.

[0044] In any optional embodiment, molecular weight of the PEG used in the present invention is 2000-5000 Da.

[0045] In another specific embodiment of the present invention, in step a), the HSA and the PFG are dissolved in an about 5-20 mM PBS solution, and in one preferred embodiment, the PBS solution is 10 mM PBS.

[0046] In a non-limited implementation, centrifugation is carried out after step a) goes on for a reaction time. In any optional embodiment, the reaction time is 10-40 hours, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 hours; in one preferred embodiment, the particular time is 15-30 hours; in one more preferred embodiment, the reaction time is 15-25 hours.

[0047] According to a non-limited implementation of the present invention, after completion of step b), filtering is performed to obtain a filtering medium, and then step c) and steps after step c) are performed.

[0048] In another implementation, centrifugation is carried out after step d) goes on for a reaction time. In any optional embodiment, the reaction time is 5-30 hours, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours; in one preferred embodiment, the particular time is 5-20 hours; in one more preferred embodiment, the reaction time is 10-15 hours.

[0049] According to a specific embodiment of the present invention, step a) reacts for 20 hours, and step d) reacts for 12 hours.

[0050] Moreover, in step c) of the present invention, the methanol and ethanol mixture is mixed according to a proportion of a weight ratio of about 7:3. In another embodiment, the methanol and ethanol mixture is mixed according to a proportion of a weight ratio of about 8:2, and in a further embodiment, the methanol and ethanol mixture is mixed according to a proportion of a weight ratio of about 6:4.

[0051] According to another embodiment of the present invention, the manufacturing method of the present invention further comprises marking a radioactive material on a surface of the nanoparticle. In a specific embodiment, the nanoparticle is added to a solution containing a radioactive material, and then the solution is filled with nitrogen for a reaction, to obtain a nanoparticle of a marked trhenium-188. It should be noted that, in the process of marking the radioactive material with the manufacturing method of the present invention, it is not necessary to use any interfacial agent as a dispersing agent or to add any stabilizer, which simplifies the conventional manufacturing procedure and significantly increases production efficiency. The nanoparticle with a radioactive material of the present invention, in addition to treating the cancer as a radioactive drug, can also diagnose diseases as a developing agent.

[0052] In any optional embodiment, the particle size of the nanoparticle of the present invention is about 1-10 nm; in one preferred embodiment, the particle size of the nanoparticle of the present invention is about 20-80 nm; and in one more preferred embodiment, the particle size of the nanoparticle of the present invention is about 50-75 nm.

[0053] Several embodiments are disclosed hereinafter to elaborate various different implementation aspects of the present invention, to enable those of ordinary skill in the art can implement the technical contents of the present invention according to the disclosure of the specification. Therefore, various embodiments disclosed hereinafter cannot be used to limit the claims of the present invention. Moreover, all documents cited in the specification can be regarded as complete reference and a part of the specification.

Example 1 Manufacturing of PEG-HSA

[0054] 316 mg of HSA and 120 mg of mal-PEG5000 (the mal ratio is 1:5) were dissolved in a 10 mM PBS solution, and reacted for 20 hours at 37 C., and after reaction, the solution was moved into an Amico Ultra-15 (50 KD) centrifuge tube, which was centrifuged for 30 minutes at a rotating speed of 4400 rpm. After centrifugation, the lower solution was removed, then 10 ml of pure water was injected to the top of the centrifuge tube, the process was repeated three times, and finally the upper solution was taken out for freeze-drying, to obtain a 254 mg sample containing mPEG-HSA.

[0055] The sample containing mPEG-HSA carried out sampling analysis with a HPLC system (a reaction condition was: RP-18 tubular column, moving phase A: 100% water 75-40% moving phase B: ACN100%+0.01% TFA 50 minutes) and sample concentration of 5 mg/ml, and retention time of mPEG-HSA was 22.51 minutes. A sample coupling rate >70% can be obtained. It can be obtained according to mass spectrum analysis that molecular weight of mPEG-HSA was about 72 Kd, and reference can be made to FIG. 1A and FIG. 1B for results.

Example 2 Manufacturing of PEG-HSA Nanoparticle

[0056] The PEG-HSA nanoparticle manufactured according to Example 1 was used to manufacture a nanoparticle, 100 mg of PEG-HSA was dissolved in a 10 mM 0.9% salt solution, after complete dissolution, a 100 uL 0.1 N sodium hydroxide solution was added slowly, and then 0.22 um filter paper was used for filtering. The filtered solution was added to a 4 ml of a methanol and ethanol mixture (a weight ratio of 7:3) through peristaltic pump, which reacted at room temperature at a rotating speed of 700 rpm. After precipitation of albumin particles, a 58.8 microlitres of 8% glutaraldehyde water solution (at a rotating speed of 700 rpm) was added slowly, then the rotating speed was reduced to 300 rpm, and the reaction went on for 12 hours. The solution after reaction was moved into the centrifuge tube and was centrifuged (30000 g, centrifuged for 30 minutes) with an ultra-high-speed vacuum centrifuge, after centrifugation, liquid supernatant was removed, after 1 ml of pure water was added to disperse sediments, centrifugation (30000 g, centrifuged for 30 minutes) was performed with a high-speed vacuum centrifuge, the process was repeated three times, to obtain a liquid suspension containing the nanoparticle of the present invention, and after analysis with a particle size analyzer, the particle size of the nanoparticle was 67.2 nm and surface potential was 25.1 mV, wherein results of the particle size were shown in FIG. 2.

Example 3 Manufacturing of the Nanoparticle of a Marked Radioactive Material of the Present Invention

[0057] In this example, the present invention selected rhenium-188 to manufacture a nanoparticle with a radioactive material. Radioactive nuclide rhenium-188 was a radioisotope with diagnosis and treatment functions, which had a moderate half life (16.9 hours) and can emit 155000 electron volts (keV) gamma rays suitable for applications to nuclear medical imaging diagnosis, and the beta energy released was up to 2.12 million electron volts (MeV), suitable for applications to nuclear medical cancer treatment.

[0058] Specific steps of the example were as follows:

[0059] A 5 mg of PEG-HSA nanoparticle was dissolved in a 500 microlitres of 0.9% sodium chloride solution. A 150 microlitres of stannous chloride solution (10 mg/ml in a 1 N hydrochloric acid solution) was added; and a 500 microlitres of citric acid solution (40 g/ml) was added. After nitrogen was filled for at least 1 minute, then a rhenium-188 solution (10 mCi/0.5 ml) was added, and after nitrogen was filled for at least 1 minute, the reaction was carried out for 60 minutes at 95 C. After the reaction ended, the mixture was cooled after 10-minute standing. After the pH value of the solution was adjusted to 5.5 with 1 N NaOH, the nanoparticle marking rhenium-188 of the present invention was obtained.

[0060] Particle size and potential of the nanoparticle marking rhenium-188 of the present invention were measured with radiochemical purity and a particle size analyzer, results showed that the chemical purity was 100%, the particle size was 64.5 nm, and the surface potential was 8.2 mV, and the results were respectively shown in FIG. 3A and FIG. 3B.

[0061] Specific examples disclosed above are not used to limit the claims of the present invention. Those of ordinary skill in the art can make modifications according to usual experience within the scope covered by the principle and spirit of the present invention, and thus the scope asserted in the present invention should be subject to that defined by the claims.