阅读说明：本技术 一种阿霉素偶联壳寡糖的纳米粒子 (Nanoparticle of adriamycin coupled chitosan oligosaccharide ) 是由 欧阳小琨 梁建浩 凌俊红 卢雨清 杨立业 于 2021-09-16 设计创作，主要内容包括：本发明利用壳寡糖(COS)和阿霉素(DOX)的氨基与苯甲醛封端聚乙二醇(DF-PEG-DF)的芳香醛自发形成pH敏感的苯亚胺键,制备得到具有pH敏感的壳寡糖载药纳米粒子(COS-DOX),使其在肿瘤弱酸性条件下实现定点释放,提高疗效和降低药物的毒副作用。(According to the invention, the pH-sensitive phenylimide bond is formed by the amino groups of Chitosan Oligosaccharide (COS) and adriamycin (DOX) and the aromatic aldehyde of benzaldehyde end-capped polyethylene glycol (DF-PEG-DF) spontaneously, so that the pH-sensitive chitosan oligosaccharide drug-loaded nanoparticle (COS-DOX) is prepared, the fixed-point release of the chitosan oligosaccharide drug-loaded nanoparticle is realized under the weak acid condition of tumor, the curative effect is improved, and the toxic and side effects of the drug are reduced.)

1. The preparation method of the adriamycin coupled chitosan oligosaccharide nano particle is characterized by comprising the following steps:

1) firstly, dissolving DOX & HCl hydrochloride in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of the DOX & HCl, adding diethylamine for mixing, desalting for 2 hours, adding a Chitosan Oligosaccharide (COS) dimethyl sulfoxide (DMSO) solution, fully stirring for 5 minutes, and slowly dropping 0.5mL of DF-PEG-DF solution under stirring to react at room temperature for 12 hours;

2) putting the reaction solution into a dialysis bag, and dialyzing in deionized water for 24h to remove unreacted DOX and micromolecules to obtain dialysis retention solution;

3) slowly adding TPP solution dropwise into the dialysis retention solution while stirring, stirring vigorously for 10min, and freeze-drying the reaction solution to obtain COS-DOX.

2. The preparation of the doxorubicin coupled chitosan oligosaccharide nanoparticle according to claim 1, wherein the weight volume ratio of doxorubicin DOX-HCL to dimethyl sulfoxide DMSO in step 1) is 1 mg/ml.

3. The preparation of the doxorubicin-conjugated chitosan oligosaccharide nanoparticle according to claim 1, wherein the molar ratio of diethylamine to doxorubicin DOX-HCL in step 1) is 2: 1.

4. The preparation of the doxorubicin conjugated chitosan oligosaccharide nanoparticle according to claim 1, wherein the concentration of the Dimethylsulfoxide (DMSO) solution of Chitosan Oligosaccharide (COS) in the step 1) is 20 mg/mL.

5. The preparation method of the doxorubicin conjugated chitosan oligosaccharide nanoparticle according to claim 1, wherein the DF-PEG-DF solution in step 1) is a 10 w/w% DF-PEG-DF aqueous solution.

6. The preparation of the doxorubicin conjugated chitosan oligosaccharide nanoparticle according to claim 1, wherein the volume ratio of the DMSO solution of DOX-HCL in step 1) to the dimethyl sulfoxide (DMSO) solution of Chitosan Oligosaccharide (COS) is 5: 4.

7. The preparation of the doxorubicin conjugated chitosan oligosaccharide nanoparticle according to claim 1, wherein the solubility of the TPP solution in the step 3) is 2 mg/L.

8. The preparation of the doxorubicin coupled chitosan oligosaccharide nanoparticle according to claim 1, wherein the volume ratio of the TPP solution in step 3) to the DMSO solution of DOX-HCL in step 1) is 1: 1.

9. An doxorubicin conjugated chitosan oligosaccharide nanoparticle prepared by the method of any one of claims 1-8.

Technical Field

The invention relates to a drug-loaded nanoparticle, in particular to an adriamycin coupled chitosan oligosaccharide nanoparticle and a preparation method thereof.

Background

pH triggered drug release is one of the commonly used strategies in drug delivery, targeting the extracellular microenvironment of tumor cells and the acidic environment of intracellular organelles. The pH-responsive delivery system can be designed for extracellular and intracellular drug release. The general pH-sensitive nano-carrier realizes drug release under the low pH condition outside or inside the cell by introducing protonatable groups, acid-labile bonds or pH-responsive 'PEG detachment' into the polymer of the carrier, thereby promoting cell uptake and intracellular drug delivery.

It has been studied to prepare drug-loaded gels as a cross-linking agent for chitosan hydrogels, however, chitosan has poor water solubility and small amounts of hydrochloric acid or acetic acid are usually added to dissolve chitosan, which increases its acid toxicity to normal tissues and limits targeted drug delivery to pH response.

Disclosure of Invention

The invention provides a preparation method of adriamycin coupled chitosan oligosaccharide nanoparticles, which utilizes amino groups of Chitosan Oligosaccharide (COS) and adriamycin (DOX) and aromatic aldehyde of benzaldehyde end-capped polyethylene glycol (DF-PEG-DF) to spontaneously form pH-sensitive phenylimide bonds to prepare the chitosan oligosaccharide drug-loaded nanoparticles (COS-DOX) with pH sensitivity.

In one aspect, the invention provides a preparation method of doxorubicin coupled chitosan oligosaccharide nanoparticles, which comprises the following steps:

1) firstly, dissolving DOX & HCl hydrochloride in dimethyl sulfoxide (DMSO) to obtain a DMSO solution of the DOX & HCl, adding diethylamine for mixing, desalting for 2 hours, adding a Chitosan Oligosaccharide (COS) dimethyl sulfoxide (DMSO) solution, fully stirring for 5 minutes, and slowly dropping 0.5mL of DF-PEG-DF solution under stirring to react at room temperature for 12 hours;

2) putting the reaction solution into a dialysis bag, and dialyzing in deionized water for 24h to remove unreacted DOX and micromolecules to obtain dialysis retention solution;

3) slowly adding TPP solution dropwise into the dialysis retention solution while stirring, stirring vigorously for 10min, and freeze-drying the reaction solution to obtain COS-DOX.

Preferably, the weight-to-volume ratio of the doxorubicin hydrochloride DOX & HCL to the dimethyl sulfoxide DMSO in the step 1) is 1 mg/ml.

Preferably, the molar ratio of diethylamine to doxorubicin DOX · HCL in said step 1) is 2: 1.

Preferably, the concentration of the Chitosan Oligosaccharide (COS) dimethyl sulfoxide (DMSO) solution in the step 1) is 20 mg/mL.

Preferably, the DF-PEG-DF solution in the step 1) is 10 w/w% DF-PEG-DF aqueous solution.

Preferably, the volume ratio of the DMSO solution of DOX & HCL to the Chitosan Oligosaccharide (COS) dimethyl sulfoxide (DMSO) solution in the step 1) is 5: 4.

Preferably, the solubility of the TPP solution in the step 3) is 2 mg/L.

Preferably, the volume ratio of the TPP solution in the step 3) to the DMSO solution of DOX & HCL in the step 1) is 1: 1.

On the other hand, the invention provides the adriamycin coupled chitosan oligosaccharide nano particle prepared by the method, and the nano particle has good biocompatibility and biodegradability, realizes fixed-point release under the weak acid condition of tumor, improves curative effect and reduces toxic and side effects of the drug.

The invention prepares the chitosan oligosaccharide nano drug-carrying particle which is pH sensitive and has good biocompatibility, biodegradation and drug delivery control, and the adriamycin coupled chitosan oligosaccharide nano particle COS-DOX prepared by the invention is an ideal drug carrier for enhancing cell uptake and curative effect.

Drawings

FIG. 1 is a schematic diagram of a synthetic method of COS-DOX.

FIG. 2 is a distribution diagram of COS-DOX 1-8 particle size in example 2.

Fig. 3 is a TEM image in example 3.

Wherein (a) is a TEM image of COS/TPP, (b) is a TEM image of COS/DOX, and (c) is a TEM image of COS-DOX/TPP.

FIG. 4 is a graph of the drug encapsulation efficiency and the loading rate of COS-DOX 1-8 in example 4.

FIG. 5 is an infrared spectrum of COS and COS-DOX 1-4 in example 5.

FIG. 6 is a thermogravimetric analysis chart of COS and COS-DOX 1-4 in example 6.

FIG. 7 is a graph of the carbon nuclear magnetic resonance spectrum of COS and COS-DOX1 in example 7.

FIG. 8 is a graph showing the release profile of COS-DOX 1-4 at different pH values in example 8.

Wherein (a) COS-DOX 1; (b) COS-DOX 2; (c) COS-DOX 3; (d) COS-DOX 4.

FIG. 9 is a graph showing the comparison of cell viability after co-culturing for 24h with HCT-116 cells at different concentrations of COS, DOX, and COS-DOX 1.

Detailed Description

The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention. The main reagents and materials used in the present invention are shown in Table 1, and the main experimental instruments are shown in Table 2.

TABLE 1 Primary reagents and materials

TABLE 2 Main Experimental Equipment

Example 1

Firstly, dissolving DOX & HCL in DMSO, preparing a DMSO solution with the solubility of 1mg/mL, adding diethylamine with the molar mass of 2 times (DOX & HCL) into the DMSO solution of DOX & HCL, mixing and desalting for 2h, adding a DMSO solution with the concentration of 20mg/mL COS, fully stirring for 5min, and slowly dropping 0.5mL of 10 w/w% DF-PEG-DF aqueous solution under stirring to react for 12h at room temperature; putting the reaction solution into a dialysis bag (MWCO: 3.5-5 kDa) and dialyzing in deionized water for 24 hours to remove unreacted DOX and micromolecules to obtain dialysis retention solution; slowly adding TPP solution (2mg/L) dropwise into the dialysis retention solution under stirring, stirring vigorously for 10min, and freeze-drying the reaction solution to obtain COS-DOX. FIG. 1 shows a preparation scheme and schematic diagram of COS-DOX, and COS-DOX 1-COS-DOX 8 are obtained according to the proportion in Table 3 and are used for subsequent experimental study.

TABLE 3

The study optimizes the particle size, drug encapsulation efficiency, loading rate and Zeta potential of nanoparticles by changing the ratio of aldehyde, amino and sodium Tripolyphosphate (TPP), and the materials were characterized by Transmission Electron Microscopy (TEM), fourier infrared spectrophotometry (FT-IR) and Thermo Gravimetric Analysis (TGA). The result shows that the particle size of the nanoparticles can be regulated and controlled by changing the proportion of each component of the adriamycin coupled chitosan oligosaccharide nanoparticles, and further the drug encapsulation efficiency and the transfer effect can be influenced. Experiments show that the COS-DOX2 nanoparticles with the minimum particle size of 168.6 +/-4.3 nm, COS-DOX1 nanoparticles with the highest potential of 35.1 +/-1.9 mV have the particle size of 219.2 +/-1.5 nm, and the drug encapsulation efficiency and the loading rate of COS-DOX1 are 5.55 +/-0.13% and 88.81 +/-2.00%. The experiment also investigated the in vitro release properties and pH responsiveness of the nanoparticles. COS-DOX and human colon cancer cells (HCT-116) are co-cultured, and the result shows that the drug-loaded nanoparticles based on COS increase the uptake of drugs by tumor cells, and remarkably enhance the cytotoxicity of DOX to HCT-116 cells within a certain concentration.

Example 2

The size and Zeta potential of COS-DOX 1-COS-DOX 8 in PBS were determined using a Zeta sizer Nano-ZS90 instrument. The morphology of the nanoparticles was observed using a FEI Tecnai G2F 20 transmission electron microscope. 2 mu L of COS-DOX solution is dripped on an ultra-thin carbon coating copper net and naturally dried for observation.

In order to explore the optimal proportion of each component of the adriamycin coupled chitosan oligosaccharide nano particle, particle size and Zeta potential analysis is carried out on the prepared COS-DOX 1-COS-DOX 8. The sizes of all the nanoparticles are between 100 and 250nm, which shows that the nanoparticles can permeate to a tumor part through a vascular gap. As can be seen from FIG. 2 and Table 4, the particle sizes of COS-DOX1, COS-DOX2, COS-DOX5, and COS-DOX6 are smaller than COS-DOX3, COS-DOX4, COS-DOX7, and COS-DOX8, mainly because more anions are introduced after more TPP is added, resulting in more COS being cross-linked and increased particle size. By comparing the particle sizes of COS-DOX1 with COS-DOX2, COS-DOX3 with COS-DOX4, COS-DOX5 with COS-DOX6, COS-DOX7 with COS-DOX8, it can be seen that the increase in the amount of COS causes an increase in the particle size, mainly due to the electrostatic interaction of ionic crosslinks that occurs primarily between COS and TPP, whereas the particle size of COS-DOX1 is smaller than COS-DOX5, the particle size of COS-DOX2 is smaller than COS-DOX6, the particle size of COS-DOX3 is smaller than COS-DOX7, and the particle size of COS-DOX4 is smaller than COS-DOX8 because the addition of increasing DOX causes more DOX to be grafted to COS. The experimental results show that the size of the final nanoparticles can be adjusted by changing the proportion of each component of the doxorubicin coupled chitosan oligosaccharide nanoparticles, which can affect the drug encapsulation efficiency and the transfer effect.

The Zeta potentials of COS-DOX 1-4 are all larger than 20mV, the Zeta potentials of COS-DOX1 and COS-DOX2 reach 35.1 +/-1.9 mV and 30.5 +/-0.4 mV, after the addition amount of TPP is increased, the potentials of COS-DOX3 and COS-DOX4 are reduced to 25.7 +/-2.5 mV and 27.3 +/-3.7 mV, mainly because TPP is introduced as an anionic cross-linking agent, more COS is cross-linked, and the surface potential is reduced, the trend can also be observed in COS-DOX 5-8, and the Zeta potentials are reduced to 20.8 +/-1.6 mV and 24.6 +/-4.3 mV of COS-DOX7 and COS-DOX8 from 29.6 +/-2.8 mV and 26.3 +/-3.2 mV of COS-DOX5 and COS-DOX 6. Therefore, the dosage of TPP in the subsequent experiment is 4 mL.

TABLE 4 particle size distribution and Zeta potential of COS-DOX 1-8 nanoparticles

Example 3

Pure COS nanoparticles (COS/TPP) of uncoupled DOX were prepared according to the ratio of COS to TPP of COS-DOX1 in Table 3, uncrosslinked COS/DOX polymer was prepared according to the ratio of COS to DOX in COS-DOX1, COS-DOX/TPP nanoparticles were prepared according to the ratio of COS, DOX, and TPP in COS-DOX1, and the change of product morphology was observed under a transmission electron microscope. As shown in fig. 3(a), the molecular chains of COS are aggregated in the presence of TPP due to the ionic crosslinking, forming COS/TPP nanoparticles. After coupling COS and DOX, a snowflake-shaped multi-branched polymer containing pH sensitive benzidine bonds is formed (FIG. 3(b)), and after adding a cross-linking agent TPP, the polymer is aggregated to form COS-DOX/TPP nanoparticles with uniform size (FIG. 3 (c)). The size and Zeta potential of successfully prepared nanoparticles of COS-DOX nanoparticles can be seen by TEM images.

Example 4

The optimal ratio of COS to DOX was examined by calculating the drug encapsulation and loading rates, and the results are shown in fig. 4. LC of COS-DOX1 and COS-DOX4 on DOX was 5.55 + -0.13% and 5.26 + -0.04%, respectively, lower than 9.61 + -0.18% and 8.55 + -0.34% of COS-DOX2 and COS-DOX3, but EE of COS-DOX1 and COS-DOX4 on DOX was 88.81 + -2.00% and 84.08 + -0.63%, higher than 76.85 + -1.43% and 68.41 + -2.72% of COS-DOX2 and COS-DOX 3. Increasing the amount of DOX added increases the LC of COS-DOX5 and COS-DOX8 to 7.62 + -0.22% and 8.07 + -0.09% for DOX, but the EE decreases to 64.80 + -1.90% and 68.63 + -0.72%, while the LC of COS-DOX6 and COS-DOX7 with low COS ratio has 10.60 + -0.56% and 10.68 + -0.40% for DOX, but the EE only has 47.71 + -2.50% and 48.07 + -1.81%, which may be that the decrease of COS ratio results in the decrease of grafting sites. Experimental results show that COS-DOX is successfully coupled with DOX, and different COS, DOX and TPP ratios can influence the drug encapsulation efficiency and the loading rate.

Example 5

The samples were tested using FT-IR, mixed with KBr pellets and ground thoroughly, tabletted and ranged from 400 to 4000cm^-1The intensity of the peak was recorded by scanning.

The infrared spectroscopy analysis was performed on COS and COS-DOX 1-4, and the results are shown in FIG. 5. About 3430cm^-1Has a broad peak of O-H and N-H stretching vibration of about 900cm^-1The doublet of radicals corresponds to-CH-and-CH₂-the presence of a group. 1634cm in infrared curve of COS^-1And 1528cm^-1The peak of (A) is due to N-H bending vibration in the amino group. The peak intensities of COS-DOX1 and COS-DOX4 were significantly higher than those of COS-DOX2 and COS-DOX3 because an increase in the proportion of COS gave DOX more grafting sites, resulting in the incorporation of more functional groups. The infrared spectra of COS-DOX 1-4 all appear to be located at 1645cm^-1This is also a characteristic absorption peak of the phenylimine bond (-C ═ N-), indicating successDF-PEG-DF grafted COS-DOX was prepared and pH responsiveness was introduced into the nanoparticles.

Example 6

Thermogravimetric analysis was performed on the raw chitosan oligosaccharide and the chitosan oligosaccharide after drug loading on a TGA 2 thermogravimetric instrument. In N₂Heating the sample at a heating rate of 10 ℃/min in an atmosphere, wherein the temperature range is 35-600 ℃.

The thermal stability of COS and COS-DOX 1-4 was examined by a re-analysis method, and the results are shown in FIG. 6. As can be seen from the COS weight loss curve, the weight loss is 6.66% below 100 ℃, the loss of physically adsorbed water and part of hydrogen bond water combined with hydroxyl and amino is mainly in the stage, the weight loss in the second stage occurs when the temperature is raised to 165 ℃, the weight loss in the stage is 38.75% at 165-300 ℃, the weight loss in the stage is caused by the complex process of sugar ring degradation and macromolecular chain disintegration in the sample, the weight loss in the stage can also be observed in the thermogravimetric curves of COS-DOX1 and COS-DOX4, and the weight loss in the COS-DOX2 and COS-DOX3 are kept stable at the temperature, because the increase of the COS proportion causes part of COS to be subjected to self-crosslinking or not grafted with DOX, and the weight loss caused by the COS degradation in one stage more than COS-DOX2 and COS-DOX 3. The maximum weight loss temperature of COS-DOX is significantly increased compared to COS, probably because the cross-linking agent binds the polymer chains together, forming hydrogen bonds, limiting their segmental motion, thus more heat energy is required for degradation. Weight loss of COS-DOX 1-4 is 46.61%, 55.21%, 45.39% and 39.24% at 355-440 ℃, and weight loss in the section is mainly oxidation of carbon and removal of oxygen-containing groups. This indicates that the coupling reduces the weight loss of COS at high temperature, indicating that the prepared COS-DOX has good thermal stability. Due to the successful grafting of DOX, the molecular structure of COS is changed, and the molecular mass, crosslinking density and interaction among molecular chains are increased, so that the thermal stability of the polymer is greatly improved.

Example 7

And performing solid nuclear magnetic detection on COS and COS-DOX by using Bruker Avance III 600M cross polarization magic angle rotation.

Solid body¹³C NMR is one of the most efficient and accurate means of determining the structure of a compound. FIG. 7 is a solid of COS and COS-DOX1¹³C NMR spectrum. Characterization of COSThe carbon signal is present at 55-75 ppm (C1:72.0ppm, C2, C6: 62.5-56.5 ppm, C3-C5: 70.7 ppm). The solid state 13C NMR spectrum of the product after DOX coupling with COS is 174.8ppm (C7: -C ═ N), 98.7-89.1 ppm (C8-C14: aromatic ring) and 24.6ppm (C15, C25: -CH)₃) A new peak appears, and the absorption peak intensity at 62.5-56.5 ppm is obviously increased, which indicates that the introduction of a new structure into the compound leads to the increase of alkyl groups. These changes were due to successful grafting of DOX, the solid phase¹³C NMR spectrum proves that the experimental method can successfully graft the DOX to the COS to prepare the COS-DOX medicine carrying particle containing the phenylimine bond.

Example 8

COS-DOX 1-4 was dispersed in PBS buffer solution of pH 7.4 and ABS buffer solution of pH 5.2, respectively, at a concentration of 0.5mg/mL, and placed in dialysis bags (MWCO: 3.5-5 kDa). The dialysis bag was placed in 100mL of the same buffer medium and released with shaking at 37 deg.C (80 rpm). At intervals, 2ml of dialysis exudate is extracted for ultraviolet visible spectrophotometry to determine DOX concentration, and meanwhile, equal buffer solution is supplemented, and the drug release rate is calculated.

Revealing the in vitro release behavior of DOX is of great significance for understanding the interaction between the drug and the carrier. A dialysis method is adopted to study the in-vitro release effect of COS-DOX 1-4. PBS (pH 7.40) and ABS (pH 5.0) were used to mimic the physiological circulation system and lysosomal environment of tumor cells, respectively. As shown in FIG. 8, the release of DOX from COS-DOX 1-4 has strong pH dependence, and the release speed of DOX is increased along with the reduction of pH value. When the pH value is 7.4, the total release rate of COS-DOX1 is 28.05 +/-1.69%, and after 18 hours, the plateau period is reached, and the release rate is about 16.17 +/-3.54%; the total release rate of COS-DOX2 is 19.90 +/-1.89%, and the release rate reaches 15.99 +/-2.55% after 12 h; the total release rate of COS-DOX3 is 17.88 +/-1.54%, and the release rate reaches about 15.16 +/-1.55% after 18 h; the total release rate of COS-DOX4 was 23.10 + -1.58%, and the release rate reached a plateau after 24h, which was about 20.00 + -2.14%. When the pH value is 5.0, the DOX in the COS-DOX 1-4 is released continuously, the accumulated release of the COS-DOX 1-4 is 93.83 +/-2.84%, 94.37 +/-1.61%, 88.40 +/-2.01% and 91.97 +/-3.48%, and the total release time reaches a plateau after about 8 hours. As can be seen from the experimental results, the release process of COS-DOX has certain pH responsiveness, which mainly depends on the phenylimide bond in the preparation, while COS-DOX3 and COS-DOX4 are not beneficial to the release of the drug because of the aggregation and the crimping caused by more cross-linking of polymer chains due to excessive TPP addition, so the release rate is lower than COS-DOX1 and COS-DOX 2.

Example 9

Cytotoxicity of COS-DOX1 on HCT-116 human colon cancer cells was determined using an MTT assay.

1) Cell recovery: the frozen HCT-116 cells are taken out and placed in a water bath kettle at 37 ℃ for quick thawing, cell sap is centrifuged at 1000rpm, cell pellets are taken out, and fresh Mccoy's 5A culture medium (containing 10 percent of fetal calf serum) is placed in a culture bottle for culture.

2) Cell culture and passage: observing the cell density in the cell culture bottle under an inverted microscope until the cell density reaches 90%, discarding the culture medium, washing with PBS for 2 times, adding 2mL of EDTA-pancreatin for digestion for 2min, adding an equivalent amount of culture medium to stop digestion, and blowing and beating uniformly to form a cell suspension.

3) Cell counting: after diluting the cell suspension, 2. mu.L of the diluted cell suspension was dropped on a cell counting plate for counting.

4) Inoculation: taking HCT-116 cells in logarithmic growth phase, inoculating the cells on a 96-well plate with the density of 6 multiplied by 10⁴Cells/well, 180. mu.L of medium per well, 37 ℃ 5% CO₂Incubate under atmosphere for 24 h.

5) Sample adding: adding 20 mu L/hole COS, COS-DOX1 and DOX with concentration of 0-100 mg/L (by DOX concentration) and culturing for 24 h.

6) And (3) survival rate testing: 20 μ L of MTT solution (5mg/mL) was added to each well and incubation continued for 4 h. After discarding the solution in the wells, 150. mu.L of DMSO was added to each well, mixed well, and the absorbance was measured at 490nm using a microplate reader. Cell viability was calculated as the percentage of absorbance of the sample wells compared to control wells that were not treated with the sample.

The MTT method was used to determine the in vitro cytotoxicity of COS, free DOX and COS-DOX1 to HCT-116 cells. As shown in FIG. 9, the inhibitory effect of COS with the concentration of 5-40 μ g/mL on cells is not obvious, and the cell survival rate of HCT-116 co-cultured with COS is still above 80% at the concentration of 40 μ g/mL, which indicates that COS has relatively low cytotoxicity. Whereas both free DOX and COS-DOX1 showed dose-dependent cytotoxicity. The survival of HCT-116 cells co-cultured with free DOX decreased from 47.05 + -2.33% to 8.13 + -0.81% and the survival of HCT-116 cells co-cultured with COS-DOX1 decreased from 39.51 + -1.71% to 8.30 + -0.71% as the dose increased from 5 μ g/mL to 40 μ g/mL. It can be seen that under the same small dose, COS-DOX1 exhibited higher cytotoxicity than DOX, which indicates that COS-based nanocarriers significantly enhanced the cytotoxicity of DOX to HCT-116 cells within a certain concentration, increased the uptake of drugs by tumors, and increased the intracellular drug delivery efficiency.

Example 10

1) Inoculation: taking HCT-116 cells in logarithmic growth phase, inoculating the cells on a 6-well plate with the density of 0.5 multiplied by 10⁶Cells/well, 1mL of medium per well, 37 ℃, 5% CO₂Incubate under atmosphere for 24 h.

2) Sample adding: 1mL of PBS, DOX, and COS-DOX1 were added to the wells at an equivalent DOX concentration of 10. mu.g/mL, and placed in CO at 37 deg.C₂Incubate in the incubator for 2, 4 and 8h, respectively.

3) Dyeing: discarding the culture solution, washing each well with PBS 3 times, adding 1 mL/well Hoechst 33258(10 μ g/mL) in dark, placing in an incubator for staining for 25min, discarding the staining agent after staining is finished, washing with PBS 3 times, and observing under a fluorescence inverted microscope.

Cell uptake and intracellular release behavior of drugs in HCT-116 cells was studied using free DOX and COS-DOX1 co-cultured with HCT-116 cells for 2, 4, and 12 h. Blue fluorescence of Hoechst 33258 was used to label nuclei, red fluorescence of DOX was used to track drugs, and the two fluorescence were mixed for study of drug distribution.

The results show that only a small amount of nanoparticles can be taken up by the cells during the first 2h, and a very small amount enters the nucleus to play a role. It is then evident that after 4h, COS-DOX1 showed a stronger red fluorescence, both in the cytoplasm and in the nucleus, indicating that more drug was phagocytosed in the cells over time, and that DOX in COS-DOX1 was better able to enter the cells, and the particles were more easily taken up by the cells and released DOX. After free DOX and COS-DOX1 were incubated with HCT-116 cells for 12h, the red fluorescence of both were enhanced more significantly, and most of the drugs entered the nucleus, but COS-DOX1 still exhibited better cellular uptake effect than free DOX, and could better exert the effect of inhibiting cancer cells. Therefore, COS-DOX1 is used as a novel carrier, has small particle size and good drug release performance, can enhance the tumor cell uptake, and can be used for targeted drug delivery.