NL2037729A - Preparation and applications of temperature-sensitive drug-loading composite nanoparticles - Google Patents
Preparation and applications of temperature-sensitive drug-loading composite nanoparticles Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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Abstract
The present disclosure belongs to the field of biochemistry, and specifically to a preparation and applications of temperature— sensitive drug—loading composite nanoparticles. In the present disclosure, a cross—linking agent genipin is first added after the 5 CN and the HBCOS are dissolved, so as to obtain the temperature— sensitive nanoparticles, and the present application has low toxicity and good biocompatibility for cells. When the obtained composite nanoparticles are mixed with a chemotherapeutic drug as a carrier to prepare the drug—loading composite nanoparticles, the 10 composite nanoparticles have good, blood compatibility and temperature sensitivity. (+ Fig.
Description
PREPARATION AND APPLICATIONS OF TEMPERATURE-SENSITIVE DRUG-LOADING
COMPOSITE NANOPARTICLES
The present disclosure belongs to the field of biochemistry, and specifically to a preparation and applications of temperature- sensitive drug-loading composite nanoparticles.
Cancer is a leading cause of death in all countries of the world, and is also an important factor in shortening life expec- tancy. Chemotherapy is an important method in the treatment of cancer. However, chemotherapeutic agents are poor in selectivity, low in drug utilization rate, and strong in toxic side effects.
Therefore, for the difference (for example, in temperatures, pH, and enzymes) between a tumor microenvironment and healthy tissue, designing a stimuli-responsive nanocarrier to improve the selec- tivity of tumor, enhance target accumulation of drugs at a tumor site, and control release becomes an important method of tumor treatment. The stimuli-responsive nanocarrier is a material having controllable contraction or expansion behaviors under specific physicochemical stimulation {including temperatures, pH, light, and ionic strength), and the nanocarrier may release embedded drugs at appropriate time and positions due to its stimuli- responsive characteristic. In various stimuli, the temperature is one of the most widely stimuli in stimuli-responsive drug delivery study, because temperature stimulation may be induced by endoge- nous or remotely-controlled thermal changes.
Many synthesized temperature responsive materials include poly{N-isopropyl acrylamide), poly(N,N-diethyl acrylamide), and poly {ethylene oxide), etc., which are developed to be applied to the field of biomedicine. However, the poor biocompatibility or biodegradability of these synthetic polymers limits the applica- tion of the polymers. Polymers derived from natural products, such as hydroxybutyl chitosan, hydroxybutyl chitosan oligosaccharide, and hydroxypropyl cellulose, also have temperature responsive characteristics, and simultaneously have good biocompatibility and biodegradability. However, the temperature sensitivity of these materials is concentration-dependent, a temperature response crit- ical point under a low concentration rises or the temperature re- sponsive characteristic disappears, such that the materials are mainly applied to smart windows, heavy metal removal, wound dress- ings, water pollution control and so on. During the application of the polymers in the field of biomedicine, the concentration will be diluted by various routes, resulting in the weakening or even disappearance of the temperature-sensitive effect of the polymers.
Therefore, carriers used for drug delivery must have diluted sta- bility, and concentration-dependent temperature-responsive poly- mers are limited in drug delivery applications.
Disadvantages of concentration-dependent temperature- responsive polymers in drug delivery applications are solved.
A stable polyelectrolyte complex is formed through an elec- trostatic interaction by using positively-charged temperature- sensitive Hydroxybutyl Chitosan (HBCOS) and negatively-charged So- dium Caseinate (CN). The CN has phosphoserine and carboxyl, and may be bonded to the positively-charged HBCOS through the electro- static interaction or load a positively-charged drug such as adri- amycin (DOX). Since the CN is easily hydrolyzed by trypsin, pep- sin, matrix metalloproteinase, cathepsin B, and the like, the CN may produce a readily biodegradable material after being recom- bined with other materials. The HBCOS and the CN are bonded through the electrostatic interaction to obtain the composite na- noparticles for cross-linking, such that stable temperature- sensitive composite nanoparticles may be obtained. The nanomateri- al may be used to embed antitumor drugs such as DOX, so as to re- alize temperature-sensitive control release of drug-loading nano- particles, thereby treating cancer.
A first aspect of the present disclosure provides a method for preparing temperature-sensitive composite nanoparticles, wherein the nanoparticles consist of Hydroxybutyl Chitosan (HBCOS) and Sodium Caseinate (CN), and is specifically prepared by the following method:
(1) respectively dissolving the CN and the HBCOS in water, mixing the CN and the HBCOS to cause a mass ratio of the CN to the
HBCOS to be 1:0.4-0.8, regulating the pH, and forming the compo- site nanoparticles; and (2) adding genipin, performing a reaction for 24~72h at 35°C- 39°C, regulating the pH, then performing dialysis for 1~2 days, and then performing freeze-drying, so as to obtain cross-linked compo- site nanoparticles.
Further, in step 2), the concentration of the CN is 0.5~1.5 mg/mL, the concentration of the HBCOS is 30-50 mg/mL, and a weight ratio of the CN to the HBCOS is 1:0.4~0.8; a pH value is 5.8-6.5.
Further, the concentration of the genipin added in step 2) is 0.01-0.1 mg/mL, and a pH is 7.0-7.8.
A second aspect of the present invention provides a method for preparing drug-loading composite nanoparticles, which are pre- pared by the following method: 1) respectively dissolving the CN and the HBCOS in water, mixing the CN and the HBCOS to cause a mass ratio of the CN to the
HBCOS to be 1:0.4-0.8, regulating the pH, and forming the compo- site nanoparticles; and 2) adding genipin, performing a reaction for 24~72h at 35°C- 39°C, regulating the pH, then performing dialysis for 1~2 days, and then performing freeze-drying, so as to obtain cross-linked compo- site nanoparticles 3) re-dissolving the composite nanoparticles prepared in step (2) with ultrapure water, regulating pH, adding a drug, performing shaking for 12~15h at room temperature under a dark condition, mo- lecular weight cut off of a dialysis membrane is 8000~12000a, then performing dialysis with the ultrapure water, and changing dialy- sate every 8~15h; and performing freeze-drying on a sample in a dialysis bag, so as to obtain the drug-loading composite nanopar- ticles.
Further, the concentration of the CN is 0.5~1.5 mg/mL, the concentration of the HBCOS is 30-50 mg/mL, and a weight ratio of the CN to the HBCOS is 1:0.4~0.8; a pH value is 5.8-6.5.
Further, the concentration of the genipin added in step 2) is
0.01-0.1 mg/mL, and a pH is 7.0-7.8.
Further, a final concentration of the composite nanoparticles dissolved in step 3) is 0.8~1.2 mg/mL; and the drug is a chemical drug.
A third aspect of the present invention is provided tempera- ture-sensitive composite nanoparticles obtained by the preparation method as claimed in any one of claims 1 to 3.
A fourth aspect of the present invention is provided drug- loading composite nanoparticles obtained by the preparation method as claimed in any one of claims 4 to 7.
The fifth aspect of the present invention is to provide for applications of the method as claimed in any one of claims 1 to 3, the method as claimed in any one of claims 4 to 7, the tempera- ture-sensitive composite nanoparticles as claimed in claim 8, or the drug-loading composite nanoparticles as claimed in claim 9 in preparation of drugs for treating tumors.
The present disclosure has the following beneficial effects:
In the present disclosure, a cross-linking agent genipin is first added after the CN and the HBCOS are dissolved, so as to ob- tain the temperature-sensitive nanoparticles, and the present ap- plication has low toxicity and good biocompatibility for cells.
When the obtained composite nanoparticles are mixed with a chemotherapeutic drug as a carrier to prepare the drug-loading composite nanoparticles, the composite nanoparticles have good blood compatibility and temperature sensitivity.
Since the CN is easily hydrolyzed by trypsin, pepsin, matrix metalloproteinase, cathepsin B, and the like, the CN may produce a readily biodegradable material after being recombined with other materials. The HBCOS and the CN are bonded through the electro- static interaction to obtain the composite nancparticles for cross-linking, such that stable temperature-sensitive composite nanoparticles may be obtained; and the composite nanoparticles may be used for embedding antitumor drugs such as DOX, so as to real- ize the temperature-sensitive control releasing of the drug- loading nanoparticles, thereby treating cancer.
Fig. 1: The temperature sensitivity of composite nanoparti-
cles: Fig. 1A shows temperature-sensitive testing of different genipin concentrations. Fig. 1B shows temperature-sensitive test- ing of nanoparticles constituted in different CN:HBCOS mass rati- os. 5 Fig. 2 shows diluted stability of composite nanoparticles.
Fig. 3 shows blood stability of composite nanoparticles: Fig. 3A shows serum stability of nanoparticles. Fig. 3B shows blood compatibility of nanoparticles.
Fig. 4 shows response release of a DOX-loading composite na- noparticles.
Fig. 5 shows anticancer activity of a DOX-loading composite nanoparticles: Fig. 5A shows cytotoxicity testing of composite na- noparticles with different concentrations; Fig. 5B shows cell sur- vival rates of the DOX-loading composite nanoparticles incubated at 37°C and 42°C for 24h, respectively.
The objectives and functions of the invention and the methods for achieving these objectives and functions will be clarified be- low by reference to exemplary embodiments. However, the present invention is not limited to the following disclosed exemplary em- bodiments; it may be implemented in different forms. The essence of the specification is merely to help those in the field to un- derstand the details of the invention.
Embodiment 1 1. Preparation of CN-HBCOS composite nanoparticles (1) CN and HBCOS were dissolved in water, 1mg/mL of the CN was mixed with the HBCOS (40mg/mL) to cause a mass ratio of the CN to the HBCOS to be 1:0.4~0.8, and pH was regulated to 6.2, so as to obtain the composite nanoparticles. (2) Genipin was added, the concentration of the genipin was 0.01~0.1 mg/mL, the reaction was performed at 37°C for 48h, the pH was regulated to 7.4, then dialysis (10000Da) was performed for 2 days, and then freeze-drying was performed, and then crosslinked composite nanoparticles were obtained. {3) Temperature-sensitive testing
The temperature sensitivity of composite nanoparticles was observed by regulating the concentration of the genipin. The re-
sults are shown in Fig. 1A: the temperature sensitivity of the composite nanoparticles was investigated by comparing changes in particle sizes of the composite nanoparticles at 37°C with 42°C; as the concentration of the genipin increased, the temperature sensi- tivity of the composite nanoparticles first increased and then re- duced; and the changes in the particle sizes are maximum when the concentration of the genipin reached 0.05 mg/mL, and the tempera- ture sensitivity was the strongest.
By regulating the mass ratio of the CN to the HBCOS, the tem- perature sensitivity of the nanoparticles was observed, and it was found that, when the concentration of the HBCOS was 0.4 mg/mL, the composite nanoparticles were not temperature-sensitive, and when the concentration of the HBCOS was 0.5 mg/mL, the composite nano- particles appeared temperature-sensitive (Fig. 1B).
Embodiment 2: Stability detection of CN-HBCOS composite nano- particles 1) Diluted stability
The stability of the composite nanoparticles (CN:HBCOS=1:0.5, the concentration of the genipin was 0.05 mg/mL) under a highly diluted condition was determined by measuring the particle sizes of the composite nanoparticles at different concentration and dif- ferent temperatures. As shown in Fig. 2, after the composite nano- particles were diluted from 1 mg/mL to 0.1 mg/mL, the particle sizes and temperature sensitivity of the composite nanoparticles all did not show significant difference. It indicated that the temperature sensitivity and stability of the composite nanoparti- cles formed through electrostatic interaction and covalent cross- linking were not affected during dilution, such that the composite nanoparticles might function in highly diluted environments. 2) Serum adsorption experiment
Anti-cancer drugs administered intravenously needed to main- tain its stability in blood. 10% of fetal bovine serum was select- ed to evaluate the stability of the composite nanoparticles in the serum, so as to prevent adsorption by non-specific protein of a drug carrier. If adsorption of a nanocarrier to serum protein oc- curs, the particle size of the nanocarrier increases, such that stability evaluation was performed by using dynamic light scatter-
ing to determine changes in particle size. The results are shown in Fig. 3A, the particle sizes of the composite nanoparticles did not increase in 3 days, such that the composite nanoparticles were stable in the presence of 10% fetal bovine serum. 3) Blood compatibility of the composite nanoparticles was further evaluated through a hemolysis test. A hemolysis rate indi- cated a degree to which a substance in contact with the blood dis- rupts the red cell membrane. If the value of the hemolysis rate was smaller, it indicated that blood compatibility of a biomateri- al was better. Red blood cells were co-incubated with the compo- site nanoparticles with different concentrations for 1h. Results were shown in Fig. 3B. Within all concentration ranges, the hemol- ysis rates of the composite nanoparticles were all negative val- ues. The results were similar to results obtained by other re- searchers using a PEG material. Therefore, it proved that the com- posite nanoparticles had good blood compatibility.
Embodiment 3 1) The composite nanoparticles prepared in Embodiment 1 were re-dissolved with ultrapure water to 1 mg/mL, the pH was regulated to 7.4, 4 mg/mL of DOX was added to make a concentration to 0.1 mg/mL, shaking was performed for 12h under a dark condition at room temperature, then dialysis was performed for 24h with 500 mL of the ultrapure water, molecular weight cut off of a dialysis bag is 10000Da, and dialysate was changed every 12h. A sample in the dialysis bag was freeze-dried, so as to obtain DOX-loading compo- site nanoparticles. 2) Entrapment rate detection
Table 1 Entrapment of DOX by composite nanoparticles “HBCOS concentration
Entrapment rate (%) Drug-loading rate (%) (mg/mL)*
O4 8254272 7624023 0.5 82.92+3.55 7.65+0.3 0.6 81.93+3.53 7.57+0.3 0.7 81.2612.84 7.51+0.24 0.8 80.06+2.44 7.41+0.2
ACN concentration was 1 mg/mL.
The composite nanoparticles had a good entrapment rate and drug-loading rate for DOX.
Embodiment 4
Temperature-sensitive release testing of drug-loading compo- site nanoparticles
Release determination was performed on the DOX-loading compo- site nanoparticles, it was found that its release kinetic curve is temperature responsive, and a releasing rate and a releasing amount at high temperatures significantly increased.
The composite nanoparticles was dissolved in a 10 mmol/L phosphate buffer (pH 7.4) to obtain a solution with a concentra- tion being 1 mg/mL, 4 mL of a sample was placed in a dialysis bag (10000Da) for dialysis, dialysate was 10 mmol/L phosphate buffer (a dialysis volume being 60 mL) with pH being 7.4 and 5.5, and di- alysis temperatures were respectively 37°C and 42°C. Samples (3 mL) were taken at 0.5, 1, 2, 3, 4, 6 and 8 hours for determination, and a determination wavelength was 485 nm. Fresh dialysate with the same volume and the same pH was added after each sampling. Re- sults were shown in Fig. 4. At 37°C, when the pH was 7.4, drugs in a composite nanocarrier was slowly released to 36.5% within 8 hours. When a temperature was heated from 37°C to 42°C, a cumula- tive release ratio within 8 hours when the pH was 7.4 was in- creased from 36.5% to 41.0%, indicating that the drug-loading com- posite nanoparticles might respond to external temperature stimu- lation, and thus being temperature-sensitive, such that the re- leasing of the drugs at higher temperatures was improved. Mean- while, at the beginning of releasing, a releasing rate of DOX was faster at 42°C. Under physiological conditions (pH 7.4), the compo- site nanoparticles carry more negative charges, and has a rela- tively-strong electrostatic interaction with the DOX, resulting in relatively-weak releasing. However, in an acidic medium expressed by tumor tissue or endosomes/lysosomes (pH 6.5-5.0), -COO- carried by the CN in the composite nanoparticles reduced, and the HBCOS started to protonate at the same time, causing a weakened electro- static interaction between the composite nanoparticles and the
DOX, thereby promoting the releasing of the DOX. At 37°C, when the pH was 5.5, the DOX in the drug-loading composite nanoparticles was released to 46.9% within 8 hours, which was significantly higher than a releasing degree at the same temperature when the pH was 7.4, proving that proton replacement under a low pH condition promoted the releasing of the DOX, thereby exhibiting pH respon- sive performance. The temperature response of the composite nano- particles was more pronounced at low pH, and when the pH was 5.5, the DOX in the drug-loading composite nanoparticles at 42°C was re- leased more rapidly, and the release within 8 hours was more thor- ough, reaching 62.0%.
Embodiment 5
DOX-loading composite nanoparticles and tumor cells were co- incubated, and it was found that antitumor activity was tempera- ture responsive, and a CCK-8 test was used to evaluate in vitro cytotoxicity of the composite nanoparticles and the DOX-loading composite nanoparticles in an HT-29 cell line of colon cancer cells.
HT-29 cells were inoculated in a 96-well culture plate at a density of 2x10°% cells per well, and cultivated for 48h. Then the cells were treated by using the composite nanocarriers with dif- ferent concentrations (2-0.125mg/mL). After 24h of incubation, 10
UL of a CCK-8 solution was added to each well, and after 1h of in- cubation, the absorbance of HBCOS-treated and untreated samples (control) was determined at 450 nm and 650 nm by using an enzyme meter. Cytotoxicity was evaluated with relative cell viability, and was 100% in a control group.
The DOX-loading composite nanoparticles were dissolved in a phosphate buffer solution with the pH being 7.4 to the concentra- tion of the embedded DOX to be 100 pg/mL. The mixture was added to a cell culture medium to make concentrations respectively 10-0.625 ng/mL, cultivation was performed for 24h in an incubator at 37°C and 42°C, respectively, and then CCK-8 was used to determine cellu- lar activity. The results are shown in Fig. 5.
Before in vitro anticancer activity of the drug-loading com- posite nanoparticles was determined, the cytotoxicity of the com- posite nanoparticles with different concentrations to the HT-29 cells of the colon cancer cells was determined first. The results are shown in Fig. 5A, after the composite nanoparticles with dif- ferent concentrations were added, the activity of all cells re- mained above 95%, it indicated that as a drug carrier, the compo- site nanoparticles had low toxicity and good biccompatibility to the cells. The cytotoxicity of the DOX-loading composite nanopar- ticles to the HT-29 cells at different temperatures was also eval- uated. Fig. 5B showed cell survival rates of the DOX-lcading com- posite nanoparticles incubated for 24h at 37°C and 42°C, respec- tively. The result shows that, the toxicity of the drug-loading nanoparticles to the HT-29 cells was obviously concentration- dependent. The cellular activity of the HT-29 cells after being incubated for 24h at 37°C and 42°C was higher than 90%, indicating that temperature itself had no inhibitory effect on cellular ac- tivity. When the concentration of the DOX was relatively low (0.625 pg/mL), the cellular activity had no obvious difference at different temperatures. When the concentration of the DOX was in- creased, the toxicity of the DOX-loading composite nanoparticles incubated at 42°C to the HT-29 cells was significantly higher than that of the drug-loading particles incubated at 37°C. This indicat- ed that when a temperature was higher than a phase transition tem- perature of a composite nanomaterial, the cytotoxicity of the DOX- loading composite nanoparticles was significantly improved. This was consistent with a release curve result of the DOX-loading com- posite nanoparticles, and the high cytotoxicity at the high tem- perature was attributed to the fact that the DOS was released more quickly from the composite nanoparticles.
A preferred embodiment of the invention is described in de- tail above, but the invention is not limited thereto. Within the scope of technical conception of the present invention, a variety of simple variations of the present invention, including various technical features combined in any other appropriate way, these simple variants and combinations shall also be regarded as the contents disclosed in the present invention and fall within the scope of protection of the present invention.
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