Preparation Of Folate-targeting Cabazitaxel Albumin Nanoparticles With High Drug Loading Based On Microfluidic Technology

Globally,the death caused by malignant tumors ranks only second to cardiovascular and cerebrovascular diseases,therefore,malignant tumors are serious threats to human health.At present,chemotherapy is one of the most commonly used methods in the clinical treatment of malignant tumors.As a new generation of semi-synthetic taxane,cabazitaxel?CTX?has a strong killing effect on a variety of malignant tumor cells,so it has wide application prospects in the treatment of malignant tumors.However,CTX itself has the disadvantages of poor solubility and lack of targeting.Therefore,in the treatment process,Tween 80 is added as a co-solvent,and severe allergic reactions are often caused.Moreover,the lack of targeting will cause great damage to the normal tissues and organs of the body,which limits the application of CTX in the treatment of malignant tumors.At present,the encapsulation of taxane chemotherapeutic drugs in albumin nanoparticles is a common strategy to improve drug solubility and reduce side effects.However,albumin nanoparticles prepared by the conventional methods may have cross-linking agents or class II organic reagents residues,which may cause potential harm to the body.At the same time,albumin nanoparticles prepared by conventional methods often have the disadvantages of wide particle size distribution,low encapsulation content of drug?EC?and lack of active targeting et.al.These problems limit the application of albumin nanoparticles in clinical practice.Microfluidic?MF?technology can precisely control the liquid flow in the micron size range,thereby improving the mixing efficiency and preparing nano formulations with uniform particle size distribution,high EC and small batch-to-batch variations.In order to reduce the toxic side effects of chemotherapy drugs and improve their tumor killing effect,this project intends to utilize human serum albumin?HSA?as a material to construct CTX-loaded nano delivery system with folate?FA?targeting based on microfluidic technology.First,albumin nanoparticles loaded with CTX?BU-NPs-CTX?without crosslinker and class II organic reagent residues were prepared via a self-assembly method.Then FA targeted nanoparticles was obtained to enhance the targeted delivery efficiency of FA-NPs-CTX.Then,based on the MF technology,the preparation method of the nanoparticles was improved,and the high-drug-loaded MF-NPs-CTX was prepared by using the inverted W-type passive mixing chip designed independently.Finally,combined with MF technology and targeted modification strategy,FA-PEG-NPs-CTX with FA targeting were prepared in order to target the delivery of chemotherapy drugs to the tumor site,thereby improving the therapeutic effect of tumors.The research of this thesis mainly includes the following contents:1.Establishment of in vivo and in vitro detection methods for CTXIn order to accurately quantify CTX,it is necessary to establish in vitro and in vivo detection methods for CTX.The in vitro detection method of CTX was established by high performance liquid chromatography?HPLC?.The results showed that the in vitro detection of CTX had good specificity,and the linear relationship was good in the range of 1.0-500.0?g/mL.At the same time,the relative standard deviation?RSD?of the the precision,accuracy,stability and sample recovery rate were all less than 3%,which meets the relevant requirements.Then,the method of detecting CTX in vivo was established by triple quadrupole mass spectrometry,and the method of detecting plasma and tissue content of CTX was established by using carbamazepine?CBZ?as internal standard.The results showed that CTX had a good specificity in plasma and tissue organs,with a good linear relationship in the range of10.0-500.0 ng/mL.The recovery of CTX in plasma samples was within 83.8%-85.7%,and the matrix effect was between 72.7%and 81.1%.In the meantime,the RSD of the precision,accuracy and stability of the detection method is less than 15%,which meets the relevant requirements.the quantification of CTX can be accurately performed.2.Preparation and evaluation of FA-targeted NPs in vitro and in vivo.In combination with the preliminary work of the laboratory,BU-NPs-CTX was prepared by HSA self-assembly method,and no cross-linking agent or class II organic reagent was introduced throughout the preparation process.Then,FA-NPs-CTX was prepared by attaching FA to the surface of BU-NPs-CTX with an amide bond.The FA content of FA-NPs-CTX was 11.96?g/mg.When incubated with 10%fetal bovine serum?FBS?for 48 h at 37°C,particle size of FA-NPs-CTX increased less than 30 nm,indicating that FA-NPs-CTX had good serum stability.Cellular uptake results showed that the uptake of FA-NPs-CTX by HeLa was 2.87 times that of BU-NPs-CTX within 4 h via FA receptor?FA?,and in the cytotoxicity evaluation experiment,FA-NPs-CTX was significantly potent against HeLa than BU-NPs-CTX under the same concentration conditions.The biosafety of FA-NPs-CTX was evaluated using the median lethal dose(LD₅₀)of mice,and the LD₅₀ of Tween-CTX was 5.68 mg/kg,while BU-NPs-CTX and FA-NPs-CTX did not show mice death at a dose of 400 mg/kg,which meant that the safe dose was increased by 70 times or more.The HeLa tumor-bearing nude mouse model was constructed,and the results of in vivo imaging and tissue distribution showed that FA-NPs-CTX could target aggregation at the tumor site.Compared with BU-NPs-CTX,the tumor growth inhibition rate of FA-NPs-CTX in HeLa tumor-bearing nude mice increased from 65.8%to 80.5%.Although there was no significant difference in tumor suppressor effect when compared with Tween-CTX,the weight loss in the Tween-CTX group was significant.In histopathological sections,severe damage to the heart,liver,lungs,and kidneys of the Tween-CTX group demonstrated strong toxic side effects of Tween-CTX.3.Preparation and evaluation of high drug loading MF-NPs-CTX properties in vitro and in vivo based on MF technology.In order to increase the EC of albumin nanoparticles and improve the drug delivery efficiency of albumin nanoparticles,MF-NPs-CTX was prepared by using self-designed inverted W-type passive mixing chip based on MF technology.The ethanol phase in which CTX was dissolved was efficiently mixed with the aqueous phase in an inverted W-type passive mixing chip,and the Reynolds of the mixing process was less than 150,which was a laminar mixing.Since the entire mixing process did not introduce crosslinking agents or class II organic reagents,MF-NPs-CTX had good biocompatibility.Through the single factor optimization method,and the encapsulation efficiency?EE?and particle size of the nanoparticles were used as indicators to optimize the preparation process of MF-NPs-CTX.Compared with BU-NPs-CTX,the EE of MF-NPs-CTX increased from 51.4%to84.3%,which increased by 64%,the EC increased from 5.7%to 15.6%,an increase of1.74 times.Meanwhile,the particle size distribution was more uniform,and the polydispersity index?PDI?<0.1.Differential scanning calorimetry results confirmed that CTX formed a new phase inside the albumin nanoparticles rather than a simple physical envelope.The hemolytic test results showed that when the concentration of MF-NPs-CTX reached to 200.0?g/mL,the hemolysis rate of red blood cells was still less than 5.0%,indicating that it had good biosafety and and was suitable for intravenous administration.The PC-3 tumor-bearing nude mouse model was constructed,and the results of in vivo imaging and tissue distribution showed that MF-NPs-CTX with uniform particle size distribution accumulated more at the tumor site.At the same time,BU-NPs-CTX with a large proportion of large particles aggregated significantly in the liver.In the tumor inhibition experiments of HeLa tumor-bearing nude mice,the tumor growth inhibition rates of Tween-CTX,BU-NPs-CTX and MF-NPs-CTX were51.4%,31.8%and 53.5%,respectively.The tumor inhibition effect of MF-NPs-CTX was significantly better than BU-NPs-CTX.4.Preparation and evaluation of folate-targeted NPs properties in vitro and in vivo based on MF technology.FA-PEG-NPs-CTX was prepared by attaching FA-PEG to the surface of MF-NPs-CTX in combination with MF technology and chemical modification.Compared with MF-NPs-CTX,the particle size of FA-PEG-NPs-CTX increased by approximately 25 nm.The morphology of FA-PEG-NPs-CTX was observed by transmission electron microscopy?TEM?.The results showed that FA-PEG-NPs-CTX were spherical and the particle size distribution was uniform.Laser confocal scanning microscopy showed that the uptake of FA-PEG-NPs-CTX by HeLa and A549 were time-dependent in 1-4 h.The results of flow cytometry showed that the uptake of FA-PEG-NPs-CTX by HeLa was 2.73 times?4 h?of MF-NPs-CTX mediated by FR.When the excess FA was added,the uptake of FA-PEG-NPs-CTX by HeLa was not significantly different from that of MF-NPs-CTX.In the HeLa tumor-bearing nude mouse model,the results of tissue distribution experiments showed that the fluorescence intensity of FA-PEG-NPs-CTX in the tumor site was significantly stronger than that of MF-NPs-CTX.Tumor growth inhibition experiment showed that the inhibition rate of FA-PEG-NPs-CTX on tumor growth was 74.1%,which was significantly higher than MF-NPs-CTX?44.2%?and Tween-CTX?59.3%?.The plasma pharmacokinetics of CTX was evaluated using SD rats,the area under the curve(AUC_last)of FA-PEG-NPs-CTX was 4.46 times that of the Tween-CTX group.In the tumor aggregation experiment of HeLa tumor-bearing nude mice,the AUC_last of FA-PEG-NPs-CTX was 1.75 times that of MF-NPs-CTX and3.43 times that of Tween-CTX,FA-PEG-NPs-CTX exhibited significant tumor-targeted aggregation effect.In this project,FA-targeted albumin nanoparticles were prepared based on MF technology,and a drug delivery system with safe,high efficiency,high EC and small batch-to-batch variation was constructed,which overcame the problem of cross-linking agent or organic reagent residues in conventional albumin nano-preparation,at the same time,it improved the targeted delivery ability of albumin nanoparticles,enhanced cytotoxicity of CTX,and achieved excellent results in malignant tumor suppression.This topic can provide reference for the preparation of nano-formulations for targeted delivery of chemotherapeutic drugs,and has a good driving effect on the clinical application of targeting albumin nano-formulations.