Patent Application
20240390868
This application claims priority to Chinese Application No. 2021111789355, filed Sep. 30, 2021. The entire disclosures of the above applications are incorporated herein by reference.
The present invention relates to the field of biomedical technology, specifically relates to a production system and method for producing nanoparticles. The related production method comprises two-phase solutions containing a photosensitizer and an anti-tumor drug combined in the combined pipeline to form a combined phase, and mixed sufficiently through a turbulent mixing device to form a stable dispersed composite nanoparticles of photosensitizer and anti-tumor drug, with a certain particle size and distribution coefficient.
The present invention belongs to the field of nano drug formulation technology.
During the treatment of tumors, there are a variety of therapeutic options to prolong the survival of patients. Anti-tumor drugs used in the treatment include chemotherapy drugs, targeted drugs, photosensitive molecules, photothermal molecules, peptides, proteins, siRNAs, etc. Among them, some drugs need to be made into nano formulations to achieve the anti-tumor effect. Commonly used nano formulations include: nanoparticles, liposomes, polymeric micelles, dendrimers, etc.
Photosensitizer/anti-tumor drug composite nanoparticles can not only play a single therapeutic effect of anti-tumor agent, but also play synergistic roles in the tumor treatment. The following photosensitizer/anti-tumor drug composite nanoparticles have been reported in the literature: Ce6-DOX composite nanoparticles (ACS Appl. Mater. Interfaces 2016, 8, 13262-13269), Ce6-HCPT composite nanoparticles (Nanoscale, 2017, 9, 14347), Ce6-SN-38 composite nanoparticles (Colloids and Surfaces B: Biointerfaces, 2020, 188,110722), ICG-PTX composite nanoparticles (J. Mater. Chem. B, 2019, 7, 6914), ICG-PTX-UA composite nanoparticles (ACS Appl. Mater. Interfaces 2017, 9, 43508), UA-LA-ICG composite nanoparticles (Acta Biomaterialia, 2018, 70, 197), SN-38/ICG composite nanoparticles (CN108159422B). Due to the complexity of the production method of such composite nanoparticles, the reproducibility of the preparation process is poor. Therefore, it is difficult to achieve scale-up and industrial production, which limits the clinical use of these photosensitizer/anti-tumor composite nanoparticles. Therefore, the continuous, large-scale and controllable production method of photosensitizer/anti-tumor composite nanoparticles is urgently needed.
The current large-scale production methods of nano formulations include high-pressure homogenization method, high-shear emulsification method, microfluidization homogenization method, etc. However, these production methods are batch preparation, which have the disadvantages of large amount of process control parameters, poor inter-batch reproducibility and difficulty in scale-up production.
Compared with the batch production methods, the continuous preparation method of nano formulations can produce uniform nanoparticles with adjustable sizes during continuous operation. Besides, the quality of nano formulations could be monitored in real time during the production process, and the nano formulations meeting the quality standards can be collected in real time.
For example, for liposome systems, continuous production can be performed using ethanol injection method. A microfluidic device of Precision NanoSystems in Canada can be used for the continuous production of liposomes (Langmuir, 2012, 28, 3633), the laminar flow of lipid organic phase and aqueous phase were rapidly mixed in a staggered mixer, and liposomes could be produced continuously by parameter optimization. However, since the microfluidic flow channel is usually in the micrometer scale, the fluid flow is in laminar conditions, the single-channel yield is extremely small, and the scale-up production requires multi-channel paralleling. The consistency of each liposome production unit needs to be controlled. The device for continuous preparation of blank liposomes using ethanol injection method reported in the literature (Pharm Res., 2016, 33, 404-416) and patent (CN107427791) can prepare blank liposomes with low polydispersity index by adjusting the flow velocity of ethanol phase and aqueous phase.
For polymer/drug nanoparticle systems, the continuous production can be achieved by flash nanoprecipitation (FNP) (U.S. Pat. No. 10,940,118B2, CN108137819, CN108542894). Nanoprecipitation method is based on the principle of kinetic control and can realize rapid production of nanoparticles by mixing turbulent fluids. This method has the characteristics of high drug loading, short production time (millisecond level), easy control of the size, easy to scale up and continuous production. The principle of preparing polymer/drug nanoparticles by nanoprecipitation method is as follows: the carrier or stabilizer (usually amphiphilic polymers) and hydrophobic drug dissolved in solvent miscible with water to form a homogeneous solution. Then, the solution is rapidly mixed with the anti-solvent (usually water) in a fixed channel. Since the hydrophobic substance is highly supersaturated in the mixed solvent, it is rapidly nucleated in water, and interacts with the polymer (usually an amphiphilic block polymer) at the same time. The polymer encapsulates the nucleating core to form polymer nanoparticles, protecting the nanoparticles from reaggregation, thereby forming nanoparticles with good aqueous dispersion (Expert Opin. Drug Deliv., 2009, 6, 865). The production devices used in the nanoprecipitation method include: confined impinging jet mixer (CIJM) (Physical Review Letters, 2003, 91, 118301; AIChE Journal, 2003, 49, 2264), multi-inlet vortex mixer, (MIVM) (Mol. Pharm., 2013, 10, 4367; Angew. Chem. Int. Ed. Engl., 2021, 60, 15590).
However, compared with liposomes and polymer nanoparticles, the microstructure of small molecule photosensitizer/anti-tumor drug composite nanoparticles is very different: liposomes have a bilayer phospholipid structure; and polymer nanoparticles are core-shell structure of hydrophobic drugs coated with amphiphilic block polymers; while small molecule photosensitizer/anti-tumor drug composite nanoparticles are self-stable photosensitizer/anti-tumor drug nanoaggregates. Therefore, whether nanoprecipitation method is suitable for the preparation of small molecule photosensitizer/anti-tumor drug composite nanoparticles is unpredictable, and many experiments are required for research, verification and optimization. So far, no reports of continuous production of photosensitizer/anti-tumor composite nanoparticles by nanoprecipitation method have been reported.
Based on the preparation of polymer/drug composite nanoparticles by nanoprecipitation method, this application has conducted repeated research and experiments to develop a preparation system for the large-scale production of photosensitizer/anti-tumor drug composite nanoparticles, which is suitable for continuous and controllable scale-up production of photosensitizer/anti-tumor drug composite nanoparticles.
The present invention is intended to overcome the defects and shortcomings of the existing photosensitizer/anti-tumor drug composite nanoparticle production technologies, and provide a production system and production method for producing nanoparticles, which is suitable for continuous scale-up production of photosensitizer/anti-tumor drug composite nanoparticles.
The present invention provides a production system for the continuous, large-scale and controllable production of photosensitizer/anti-tumor drug composite nanoparticles, which is suitable for the production of nano formulations including but not limited to, nano formulations in the particle size range of 1-1000 nm, and the nano formulations are photosensitizer/anti-tumor drug composite nanoparticles.
The present invention provides a (continuous, large-scale and controllable) production system (for producing photosensitizer/anti-tumor drug composite nanoparticles), which includes (1) a first pipeline, (2) a second pipeline, (3) a combined pipeline and its (fluid) outlets;
Wherein, the first pipeline and the second pipeline are connected to the combined pipeline, the first phase solution enters the combined pipeline through the first pipeline outlet, the second phase solution enters the combined pipeline through the second pipeline outlet, and the first phase solution and the second phase solution are mixed in the combined pipeline to form a combined phase; the combined phase flows out through the outlet of the combined pipeline.
In some embodiments, the core part of the production system includes: (1) a first pipeline; (2) a second pipeline; (3) a combined pipeline; (4) turbulent mixing device; (5) fluid outlet;
Wherein, the first pipeline and the second pipeline are connected to the combined pipeline, the first phase solution enters the combined pipeline through the first pipeline outlet, the second phase solution enters the combined pipeline through the second pipeline outlet, the first phase solution and the second phase solution are mixed in the combined pipeline to form a combined phase. The combined phase is fully mixed by a turbulent mixing device. After the mixing, the composite nanoparticles are collected into a suitable container through the outlet of the combined pipeline.
In some embodiments, the first pipeline is coaxial with the combined pipeline and the second pipeline is perpendicular to the combined pipeline.
In some embodiments, the mixing is turbulent mixing. The turbulent mixing can be achieved by adding a turbulent mixing device in the combined pipeline. There can be one or more turbulent mixing devices.
In some embodiments, the first pipeline outlet is positioned within the combined pipeline.
In some embodiments, the first pipeline outlet is a spray hole with a certain shape and diameter, and the first phase solution passes through the first pipeline and enters the combined pipeline through the spray hole.
In some embodiments, the spray hole diameter D1(S) at the end of first pipeline is selected from 0.03-5.0 mm; the second pipeline inner diameter D2(IN) is selected from 0.3-50.0 mm; the combined pipeline inner diameter D3(IN) is selected from 0.3-50.0 mm.
In some embodiments, the combined pipeline length (i.e., combined phase length) is selected from 6 to 120 cm, such as 9 cm.
In some embodiments, the ratio of the combined pipeline length to the combined pipeline inner diameter is (16-17): 1, such as 16.7:1.
In some embodiments, the first pipeline outer diameter D1(O) is 2 mm.
In some embodiments, the spray hole diameter D1(S) at the end of first pipeline is 0.3 to 0.6 mm, such as 0.3 mm or 0.6 mm.
In some embodiments, the second pipeline outer diameter D2(O) is 6 mm.
In some embodiments, the second pipeline inner diameter D2(IN) is 5.4 mm.
In some embodiments, the combined pipeline outer diameter D3(O) is 6 mm.
In some embodiments, the combined pipeline inner diameter D3(IN) is 5.4 mm.
In some embodiments, the second pipeline inner diameter D2(IN) is the same as the combined pipeline inner diameter D3(IN).
In some embodiments, the ratio of the spray hole diameter D1(S) at the end of first pipeline to the combined pipeline inner diameter D3(IN) can be 1:(2-50), such as 1:9 or 1:18.
In some embodiments, the turbulent mixing device is a device that mixes the first phase solution and the second phase solution to reach a turbulent flow state, such as a static mixer. The static mixer can be selected from one or more of SV type static mixer, SX type static mixer, SL type static mixer, SH type static mixer and SK type static mixer, preferably SK type static mixer.
In some embodiments, the materials used in the first pipeline, the second pipeline, the combined pipeline, the turbulent mixing device, and the fluid outlet are each selected from one or more of stainless steel, polytetrafluoroethylene, polyethylene, polypropylene, latex, silicone, or other polymer materials.
In some embodiments, the turbulent mixing can cause the fluid in the combined phase to reach a turbulent transition state or turbulent flow state by increasing the fluid flow velocity. The Reynolds number in the combined phase depends on the smoothness of the circular stainless steel pipe wall. For example, when the pipe wall is rough, a lower Reynolds number can also achieve turbulent mixing conditions (such as Re between 500-2000, this range is usually considered to be a laminar flow conditions).
In some embodiments, the turbulent mixing can be achieved by making the combined phase a bent pipe with a certain curvature, by changing the direction of fluid flow, enhancing the convection of the fluid, and enhancing the mixing of the fluid. At this time, in addition to being greater than 4000, the Reynolds number in the combined phase calculated based on the fluid in the circular tube can also be between 500-4000.
In some embodiments, the turbulent mixing can be achieved by equipping a static mixer in the combined phase. The static mixer can include but is not limited to: SV type static mixer, SX type static mixer, SL type static mixer, SH type Static mixers, SK type static mixers, etc. that can divide the fluid through turbulent mixing elements, change the flow direction of the fluid, enhance the convection of the fluid, and increase the mixing of the fluid. Under these circumstances, in addition to being greater than 4000, the Reynolds number in the combined phase calculated based on the fluid in the circular tube can also be between 500-4000.
In some embodiments, the quantity of flow Q1 of the first phase solution through the first pipeline is selected from 1-1000 ml/min; wherein the temperature T1 of the first phase solution is selected from 0-90° C.; the quantity of flow Q2 of the second phase solution through the second pipeline is selected from 10-10000 ml/min; the temperature T2 of the second phase solution is selected from 0-90° C.
The present invention also provides a (continuous, large-scale and controllable) production method for producing photosensitizer/anti-tumor drug composite nanoparticles, which includes the following steps: In a production system as described above, a first phase solution and a second phase solution are mixed, and the resulting photosensitizer/anti-tumor drug composite nanoparticles are collected from combined phase through the fluid outlet;
The solvent in the first phase solution is a good solvent for an anti-tumor drug or its pharmaceutically acceptable salt, and the solute is (1) an anti-tumor drug or its pharmaceutically acceptable salt, and a photosensitizer, or (2) an anti-tumor drug or its pharmaceutically acceptable salt;
The solvent in the second phase solution is an anti-solvent of the anti-tumor drug or its pharmaceutically acceptable salt, and the solute is (1) absent, or (2) a photosensitizer;
When the solute in the first phase solution is an anti-tumor drug or its pharmaceutically acceptable salt, and a photosensitizer, the solute in the second phase solution does not exist;
When the solute in the first phase solution is an anti-tumor drug or its pharmaceutically acceptable salt, the solute in the second phase solution is a photosensitizer.
In some embodiments, the production methods include:
Wherein, during the mixing process of the first phase solution and the second phase solution in the combined phase, under the action of turbulent shear, the two-phase solutions are rapidly mixed. The anti-tumor drug or its pharmaceutically acceptable salt dissolved in the first phase solution reaches a highly supersaturated state in the combined phase, and undergoes rapid nucleation in the mixed solvent. The core of anti-tumor drug or its pharmaceutically acceptable salt grows while interacting with the photosensitizer to form a composite nano formulation of photosensitizer and anti-tumor drug that is stably dispersed in the mixed solvent of the first phase and the second phase with a certain particle size and distribution coefficient. The obtained photosensitizer and anti-tumor drug composite nano formulation is further purified by ultrafiltration to remove pharmaceutically unusable solvents, added a freeze-drying protective agent, filtered, sterilize, filled and freeze-dried under aseptic conditions to obtain different clinically available nano formulations.
In some embodiments, the production method includes:
In some embodiments, the temperature of the first phase solution is 0-90° C., such as 25° C.
In some embodiments, the temperature of the second phase solution is 0-90° C., such as 25° C.
In some embodiments, the fluid Reynolds number Re of the combined phase is 800 to 7700, such as 839, 1934, 2579, 3868, 5158, 5236, 5539, 5841, 5236, 6477, 6750, 7053, 7356 or 7659), preferably 3000 to 7700, more preferably 3868 to 7659.
In some embodiments, the flow velocity ratio FVR between the first phase solution and the combined phase is 5 to 26 (e.g., 5.2, 5.8, 17.3, 18.1, 18.9, 19.8, 20.8, 21.9, 23.1, 24.4 or 26), preferably 17 to 26, more preferably 20.8 to 26.
In some embodiments, when the Re of the combined phase is less than 3000, the FVR of the production system is 17 to 26, and/or the production system further includes a static mixer.
In some embodiments, when the Re of the combined phase is less than 3868, the FVR of the production system is 20.8 to 26, and/or the production system further includes a static mixer.
In some embodiments, when the FVR of the production system is less than 17, the Re of the combined phase is 3000 to 7700, and/or the production system further includes a static mixer.
In some embodiments, when the FVR of the production system is less than 20.8, the Re of the combined phase is 3868 to 7659, and/or the production system further includes a static mixer.
In some embodiments, the photosensitizer includes one or more of IR780, IR820, indocyanine green or indocyanine green analogs; the porphyrin type molecules are selected from hematoporphyrin monomethyl ether; the porphyrin precursor is selected from one of 5-aminolevulinic acid and/or 5-aminolevulinic acid esters; the phthalocyanine type molecules are selected from copper phthalocyanine, cobalt phthalocyanine, aluminum phthalocyanine, nickel phthalocyanine, calcium phthalocyanine, sodium phthalocyanine, magnesium phthalocyanine, zinc phthalocyanine, indium phthalocyanine, oxytitanium phthalocyanine, manganese phthalocyanine, or one or more of the phthalocyanine derivatives; the chlorin type molecules are selected from one or more of chlorin, talaporfin, verteporfin, temoporfin, rostaporfin, porfimer sodium, hemoporfin and HPPH.
In some embodiments, the photosensitizer is selected from one or more of cyanine type molecules, porphyrin type molecules, porphyrin precursors, phthalocyanine type molecules and chlorin type molecules; wherein, the cyanine type molecules are preferably selected from one or more of indocyanine green (IR780), new indocyanine green (IR820), indocyanine green and indocyanine green analogs; the porphyrin type molecule is preferably selected from hematoporphyrin monomethyl ether; the porphyrin precursor is preferably selected from 5-aminolevulinic acid and/or 5-aminolevulinic acid esters; the phthalocyanine type molecule is preferably selected from one or more of copper phthalocyanine, cobalt phthalocyanine, aluminum phthalocyanine, nickel phthalocyanine, calcium phthalocyanine, sodium phthalocyanine, magnesium phthalocyanine, zinc phthalocyanine, indium phthalocyanine, oxytitanium phthalocyanine, manganese phthalocyanine or phthalocyanine derivatives; the chlorin type molecules are preferably selected from one or more of chlorins, talaporfin, verteporfin, temoporfin, rostaporfin, porfimer sodium, hemoporfin and HPPH.
In some embodiments, the photosensitizer is indocyanine green or chlorin e6.
In some embodiments, the anti-tumor drug contains one or more of aromatic rings or aromatic heterocycles in its structure. The anti-tumor drugs are selected from one or more of camptothecin type compounds, paclitaxel type compounds, anthracycline type compounds, targeted drugs or other anti-tumor drugs; wherein the camptothecin type molecules are preferably selected from one or more of camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, lurtotecan, gimatecan, belotecan, 10-hydroxycamptothecin, 10-hydroxy-7-ethyl-camptothecin (SN-38), exatecan, topotecan and deruxtecan; the paclitaxel type compounds are preferably selected from one or more of paclitaxel, docetaxel, cabazitaxel, 7-epipaclitaxel, 2′-acetylpaclitaxel, 10-deacetylpaclitaxel, 7-epi-10-deacetyltaxol, 7-xylosyltaxol, 10-deacetyl-7-glutarylpaclitaxel, 7-N,N-dimethylglycylpaclitaxel, 7-L-alanylacetaxel and larotaxel; the anthracycline type compounds are preferably selected from one or more of doxorubicin, epirubicin, daunorubicin, pirarubicin and aclacinomycin; the targeted drugs are preferably selected from one or more of gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, osimertinib, imatinib, sorafenib, sunitinib, lapatinib, dasatinib, olaparib, niraparib, rucaparib, fluzoparib, pamiparib, veliparib, talazoparib and apatinib; the other anti-tumor drugs are preferably selected from one or more of etoposide, teniposide, vinblastine, vincristine, vinorelbine, vindesine, maytansine, curcumin, harringtonine, homoharringtonine, gemcitabine, capecitabine, fludarabine, cladribine, pemetrexed, bortezomib, carfilzomib, ixazomib, carmustine, fluorouracil, cytarabine, cyclosporine A, eribulin and trabectedin.
In some embodiments, the anti-tumor drug is camptothecin, 10-hydroxycamptothecin, exatecan, Dxd, paclitaxel, sorafenib or curcumin.
In some embodiments, in the photosensitizer/anti-tumor drug composite nanoparticles, the combination of the photosensitizer and the anti-tumor drug is a combination of indocyanine green and camptothecin, indocyanine green and 10-hydroxycamptothecin, indocyanine green and 7-ethylcamptothecin, indocyanine green and 7-ethyl-10-hydroxycamptothecin, indocyanine green and exatecan, indocyanine green and Dxd, indocyanine green and paclitaxel, indocyanine green and docetaxel, indocyanine green and cabazitaxel, indocyanine green and sorafenib, indocyanine green and curcumin or chlorin e6 and 7-ethyl-10-hydroxycamptothecin;
The preferable combination of the photosensitizer and the anti-tumor drug is a combination of indocyanine green and camptothecin, indocyanine green and 10-hydroxycamptothecin, indocyanine green and exatecan, indocyanine green and Dxd, indocyanine green and paclitaxel, indocyanine green and sorafenib, indocyanine green and curcumin, indocyanine green and 7-ethyl-10-hydroxycamptothecin or chlorin e6 and 7-ethyl-10-hydroxycamptothecin;
In some embodiments, the range of anti-tumor drug/(anti-tumor drug+photosensitizer) in the composite nanoparticles is 0.1-0.9, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%.
In some embodiments, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (1-15):1, such as 1:1, 2:1, 5:1, 6:1, 7:1, 8:1, 10:1 or 15:1.
In some embodiments, when the anti-tumor drug is 7-ethyl-10-hydroxycamptothecin and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (2-15):1, such as 2:1, 5:1, 10:1 or 15:1.
In some embodiments, when the anti-tumor drug is camptothecin and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (1-10):1, such as 1:1, 2:1, 5:1 or 10:1.
In some embodiments, when the anti-tumor drug is 10-hydroxycamptothecin and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (1-10):1, such as 1:1, 2:1, 5:1 or 10:1.
In some embodiments, when the anti-tumor drug is exatecan and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (2-15):1, preferably (2-10):1, such as 2:1, 5:1 or 10:1.
In some embodiments, when the anti-tumor drug is Dxd and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (5-10):1, such as 5:1 or 10:1.
In some embodiments, when the anti-tumor drug is sorafenib and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (6-8):1, such as 6:1 or 8:1.
In some embodiments, when the anti-tumor drug is paclitaxel and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (1-10):1, such as 1:1, 2:1, 5:1 or 10:1.
In some embodiments, when the anti-tumor drug is curcumin and the photosensitizer is indocyanine green, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is (5-8):1, such as 5:1, 6:1, 7:1 or 8:1.
In some embodiments, when the anti-tumor drug is 7-ethyl-10-hydroxycamptothecin and the photosensitizer is chlorin e6, the molar ratio of the anti-tumor drug or its pharmaceutically acceptable salt to the photosensitizer is 2:1.
In some embodiments, the solvent used in the first phase solution and the second phase solution is water, an aqueous buffer solution with a certain pH value, or an organic solvent miscible with water. Further, the organic solvent is one or more of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol, DMF, DMAc, N-methylpyrrolidone, DMSO, butyl sulfone, tetramethylene sulfone, THF, 2-methyltetrahydrofuran, acetonitrile, acetone, ethylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, HMPA, dioxane, formic acid, acetic acid, hydroxypropionic acid, ethylamine, ethylenediamine, glycerol or pyridine.
In some embodiments, the solvent in the first phase solution is selected from one or more of methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol, DMF, DMAc, N-methylpyrrolidone, DMSO, butyl sulfone, tetramethylene sulfone, THF, 2-methyltetrahydrofuran, acetonitrile, acetone, ethylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, HMPA, dioxane, formic acid, acetic acid, hydroxypropionic acid, ethylamine, ethylenediamine, glycerol and pyridine, preferably DMSO.
In some embodiments, the solvent in the second phase solution is water or a buffer with a pH of 2-10, preferably water.
In some embodiments, the molar concentration of the anti-tumor drug or its pharmaceutically acceptable salt in the first phase solution is 0.01-0.3M; preferably 0.05-0.1M, such as 0.05M or 0.1M.
In some embodiments, the molar concentration of the photosensitizer in the first phase solution or the second phase solution is 0.01-0.3M; preferably 0.05-0.1M, such as 0.05M or 0.1M.
In some embodiments, the concentration of the anti-tumor drug in the first phase solution ranges from 0.1 to 200 mg/ml, for example, it can be 0.1 mg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, 180 mg/ml, 200 mg/ml, preferably 10-100 mg/ml.
In some embodiments, the concentration of the photosensitizer in the first phase solution or the second phase solution ranges from 0.1 to 200 mg/ml, for example, it can be 0.1 mg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 20 mg/ml, 40 mg/ml, 60 mg/ml, 80 mg/ml, 100 mg/ml, 120 mg/ml, 140 mg/ml, 160 mg/ml, 180 mg/ml, 200 mg/ml, preferably 10-100 mg/ml.
In some embodiments, the production method also includes the following post-reaction processing steps: adding water to the prepared mixed solution and ultrafiltration; or further concentration and ultrafiltration.
Wherein, the ultrafiltration can use a 2-100 kDa ultrafiltration membrane, preferably a 30 kDa ultrafiltration membrane.
In some embodiments, nanoparticles are prepared with an (average) particle size of less than 1000 nm.
In some embodiments, nanoparticles are prepared with an (average) particle size of less than 500 nm.
In some embodiments, nanoparticles are prepared with an (average) particle size of less than 200 nm.
In some embodiments, nanoparticles are prepared with an (average) particle size of 50-200 nm.
In some embodiments, nanoparticles are prepared with a polydispersity index of less than 0.3.
In some embodiments, nanoparticles are prepared with a polydispersity index of less than 0.2.
In some embodiments, nanoparticles are prepared with a polydispersity index of less than 0.1.
In some embodiments, the SN-38 encapsulation efficiency of the prepared SN-38/ICG nanoparticles in the 15-20 ml scale is 98.2%, and the nanoparticle diameter is 114±6 nm; the SN-38 encapsulation efficiency in the 60 ml scale is 91.7%, and the particle size of the nanoparticles is 127±6 nm; the encapsulation efficiency of SN-38 in the 557 ml scale is 89.2%, and the particle size of the nanoparticles is 129±10 nm; the encapsulation efficiency of SN-38 can be maintained to be greater than 80% during the scale-up process.
The present invention also provides a photosensitizer/anti-tumor drug composite nanoparticle, which is prepared by the above production method.
Compared with the prior art, the present invention has the following advantages:
The abbreviations used in the present invention are shown in Table 1.
The corresponding structures of materials used in the present invention are shown in Table 2.
The present invention is further described below by examples, but the present invention is not limited to the scope of the described examples.
Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.
The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values close to such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These numerical ranges shall be deemed to be specifically disclosed herein.
The turbulent mixing part in this application is a circular pipe with a certain diameter and length, and turbulent flow conditions are achieved through one or more of the following methods:
Increase flow velocity:
Wherein, Re is the Reynolds number, Q is the quantity of flow, d is the pipeline diameter, υ is the fluid flow velocity in the pipe, μ is the fluid viscosity, 20° C. μwater=10−3 Pa·s
It should be pointed out that for the preparation of certain composite nanoparticles of photosensitizers and anti-tumor drugs, nano-formulations with a certain particle size and particle size distribution can also be obtained with Re in the range of 500-4000.
Change the shape of the pipeline:
By increasing the tortuosity of the pipeline, the direction of fluid flow is forcibly changed, which enhances the fluid mixing.
Add a static mixer to the pipeline:
The static mixer can include but is not limited to: SV type static mixer, SX type static mixer, SL type static mixer, SH type static mixers, SK type static mixers, etc. that can divide the fluid through turbulent mixing elements, change the flow direction of the fluid, enhance the convection of the fluid, and increase the mixing of the fluid.
The SV type static mixer unit is a cylinder assembled from certain regular wave plates.
The SX type static mixer unit consists of many X-shaped units composed of crossed horizontal bars according to certain rules.
The SL type static mixer unit is composed of crossed horizontal bars according to a certain pattern to form a single X-shaped unit.
The SK type static mixer unit is composed of single-channel left and right twisted spiral plates welded together.
SH type static mixer unit is composed of double channels, with a fluid redistribution chamber between the units.
Preparation of the first phase solution: ICG 1.30 g and SN-38 1.30 g were dissolved in 35.1 g DMSO, total weight 37.7 g, SN-38 content: 3.4 wt. %, ICG content: 3.4 wt. %, SN-38 and ICG molar ratio is 2:1.
Unless otherwise specified, the second phase solution is water.
Instrument: Agilent1260 HPLC
Column: Waters XBridge C18 4.6*150 mm, 3.5 μm
Mobile phase: Use 10 mmol/L sodium dihydrogen phosphate solution (adjust to pH 4.0 with phosphoric acid) as phase A, use acetonitrile as phase B, and perform gradient elution according to the following table:
Chromatographic parameters: flow rate: 1 ml/min; column temperature: 35° C.; detection wavelength: 264 nm; injection volume: 10 μl
Diluent: DMSO
Test solution: Use a pipette to accurately take 200 μl of the SN-38/ICG nano-formulation solution, place it in a 10 ml volumetric flask, add DMSO to dissolve and quantitatively dilute to the mark and shake well.
Reference substance solution: Take about 10 mg of ICG or SN-38 reference substance, weigh it accurately, put it in a 100 ml volumetric flask, add DMSO to dissolve it and quantitatively dilute it to the mark and shake well.
Calculation method: Peak area external standard method
Unless otherwise specified, all other compounds are measured using this mobile phase, and the wavelength depends on the specific compound.
Dynamic light scattering method: The concentration of nanoparticles is 10-100 μg/ml, and the particle size and distribution of nanoparticles are measured with a nanoparticle sizer (laser light source 633 nm). Each sample is measured three times, and the average and variance of the nanoparticle size are calculated. The particle size distribution of different nanoparticles is shown in
Since the photosensitizer molecules usually absorb the laser light source (633 nm) of the dynamic light scattering instrument to a certain extent, the intercept in the fitting diagram is low, and there is a certain deviation between the three measurement results of the same sample. Therefore, the average of the three measurements is used to reduce the deviation. The peaks in the micron range in the figure are usually caused by dust.
Take 1 ml of SN-38/ICG solution, filter with a 0.22 μm nylon needle filter, and measure the SN-38 concentration by HPLC.
Unless otherwise specified, the encapsulation efficiency of hydrophobic drugs was measured according to this method.
Calculated based on the fluid in the circular pipeline, the combined phase Re=839, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re-1934, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=2579, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=3868, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5158, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=839, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=1934, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=2579, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=3868, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5158, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=839, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=1934, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=3868, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5158, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=6477, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re-839, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re-1934, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=3868, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re-5158, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re-5236, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5539, with no static mixer.
Inner diameter of the combined pipeline D3(IN)=5.4 mm;
Calculated based on the fluid in the circular pipeline, the combined phase Re=5841, with no static mixer.
Spray hole diameter at the end of first pipeline D1(S)=0.3 mm;
Calculated based on the fluid in the circular pipeline, the combined phase Re=5236, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=6750, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=7053, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=7356, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=7659, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5841, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5841, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with no static mixer.
Calculated based on the fluid in the circular pipeline, the combined phase Re=6477, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
Calculated based on the fluid in the circular pipeline, the combined phase Re=5158, with SK type static mixer, static mixer size: 5.3 mm*85 mm, with a total of 16 repeating spiral plates.
The SN-38 concentration of the preparation solution was 2.05 mg/ml, the SN-38 concentration after filtration of 0.22 μm membrane was 1.89 mg/ml, the encapsulation efficiency of SN-38: 92.2%, the original solution was added 871.4 g water and purified with 0.05 m230 kDa PES ultrafiltration membrane.
Ultrafiltrate was concentrated to 166.1 g, SN-38 concentration was 5.07 mg/ml; After filtration with 0.22 μm membrane, the SN-38 concentration was 4.60 mg/ml, and the encapsulation efficiency of SN-38 after ultrafiltration was 90.8%.
Particle size change before and after ultrafiltration:
SN-38 was dissolved in DMSO (39.2 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), SN-38 in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with no static mixer.
Camptothecin was dissolved in DMSO (20 mg/ml, 0.057 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), camptothecin in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re=6477, with no static mixer.
10-Hydroxycamptothecin was dissolved in DMSO (36.4 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), 10-Hydroxycamptothecin in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with no static mixer.
Exatecan (free base, Mw=435.4) was dissolved in DMSO (43.5 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), Exatecan in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re-6477, with no static mixer.
Dxd (Mw=493.5) was dissolved in DMSO (49.4 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), Dxd in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re=6477, with no static mixer.
Sorafenib was dissolved in DMSO (46.5 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), Sorafenib in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
PTX was dissolved in MeOH (42.7 mg/ml, 0.05 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), PTX in MeOH and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
Curcumin was dissolved in DMSO (36.8 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), Curcumin in DMSO and ICG in DMSO were mixed in proportion following the table below as the first phase solution, and water as the second phase solution.
After centrifugation, the solution with a volume ratio of 2:1 and 4:1 was clear, and no precipitate was found. The precipitation gradually increased from 5:1.
Ce6 was dissolved in DMSO (59.7 mg/ml, 0.1 M), SN-38 was dissolved in DMSO (39.2 mg/ml, 0.1 M), SN-38 in DMSO and Ce6 in DMSO were mixed in 2:1 volume ratio as the first phase solution, and water as the second phase solution.
Calculated based on the fluid in the circular pipeline, the combined phase Re=1934, with no static mixer.
15.72 mg paclitaxel and 36.42 mg ICG were added to a 50 ml eggplant flask and dissolved in 5 ml methanol. The paclitaxel content was −30 wt. %. The methanol was removed in vacuo. Adding 7.860 ml of deionized water pre-warmed at 60° C. and rotating hydration at 60° C. did not yield uniform PTX/ICG nanoparticles.
The published literature shows that mPEG2k-PLA2k and PTX can form PTX/mPEG-PLA micelles at −20 nm by thin-film hydration method with a drug load of 30 wt. % and an encapsulation efficiency of >90%.
Therefore, the properties of polymers and photosensitizers are fundamentally different and cannot be simply replaced.
SN-38 was dissolved in DMSO (39.2 mg/ml, 0.1 M), ICG was dissolved in DMSO (77.5 mg/ml, 0.1 M), SN-38 in DMSO and ICG in DMSO were filtered with 0.22 μm membrane and mixed in proportion following the table below in 1.5 ml EP tube.
Water was pre-added to 8 ml vials according to the following table, placed in a 120 W ultrasonic bath, SN-38 and ICG DMSO mixture was quickly added to the water with a pipette, and continued to sonicate for 30 s to determine the particle size distribution and encapsulation efficiency.
When the molar ratio of SN-38 to ICG was 10:1-1:1, the encapsulation efficiency of SN-38 decreased from 72.3% to 27.1%, and the encapsulation efficiency of SN-38 was only increased to 76.1% from 36.9% with 300 W probe sonicator (SN-38:ICG=2:1).
Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only illustrations, and that various changes or modifications may be made to these embodiments without deviating from the principle and substance of the present invention. Therefore, the scope of protection of the present invention is limited by the attached claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111178935.5 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/123347 | 9/30/2022 | WO |
