In this experiment, Nab-PTX-PA was prepared by using chloroform and anhydrous ethanol as the solvent, emulsified and homogenized under high-pressure homogenization without using the surfactant. The operation process was simple, and the liposolubility of PTX-PA was stronger than PTX. During the preparation process, albumin was not easy denatured at low temperature, and finally the organic solvent was removed by ultrafiltration. PTX-PA was more stable in an amorphous state and not easy to leak during storage, thus improving the stability of the product. The prepared Nab-PTX-PA was spherical and evenly distributed, with a particle size of 87.63 ± 1.15 nm (n = 3), a single peak, PDI = 0.185 ± 0.009 (n = 3), and Zeta potential of − 11.70 ± 0.61 mV (n = 3). It was found that the drug loading could be twice large as Abraxane® (20 mg/kg). The result of the experiment with Nab-PTX-PA in vitro release and the degradation of PTX-PA into PTX showed that Nab-PTX-PA could release PTX-PA from plasma and transform into active mother drug PTX under the action of enzyme ester in vivo, which is consistent with the expectation of the experiment. After conversion to the mother drug, the activity of 2-OH was restored, which provided the possibility to achieve the anti-tumor effect. PTX was Modified to PTX-PA by one step esterification, the more fat-soluble, the stronger it bound within hydrophobic pockets of albumin, and the steric hindrance of the ester group increases, which made hydrolysis difficult, and the main purpose of preparing Nab-PTX-PA was to slow down the release.
The ability of drug uptake by cells is an important index to reflect the efficacy. The results of cell uptake experiments showed that Nab-PTX-PA could be ingested by 4T1 tumor cells and had a potential anti-tumor effect. The result of cell proliferation toxicity in vitro showed that the Nab-PTX-PA had concentration-dependent toxicity, and the cell proliferation toxicity was significantly lower than that of the commercially available Abraxane®. On the basis of in vitro experiments, we performed a tissue distribution experiment and a pharmacodynamics study to investigate bone marrow suppression of the testing drugs. The result of the tissue distribution experiment showed that the concentration of PTX metabolized by Nab-PTX-PA increased slowly in the tumor site, which proved that Nab-PTX-PA could be continuously transformed into PTX to increase its accumulation in the tumor site. As a result, the anti-tumor potential was increased. After intravenous injection of Abraxane®, the drug concentration in the liver and kidney was relatively high, and the drug concentration decreased rapidly in various tissues of the mice, and the same trend was also observed in the tumor site, indicating that the time of the anti-tumor action of Abraxane® was shortened after it entered the body. After injection of Nab-PTX-PA into the tail vein, the concentration of PTX-PA distribution was increased in all tissues except the tumor site, indicating a wide range of PTX-PA distribution, but the prodrug itself was not active. So it was speculated that PTX-PA had relatively small damage to the normal organs, and the concentration of metabolized PTX increased gradually in various tissues, with the liver and lung predominating, which is consistent with the general characteristics of tissue distribution of NPs and has proved to have no special toxicity. At the tumor site of the tumor-bearing mice, the metabolic PTX concentration increased gradually, which is more beneficial to the anti-tumor effect in the tumor site for a long time. The results of pharmacodynamic study in vivo showed no significant difference in the anti-tumor effect between Nab-PTX-PA (25.58 mg/kg) and Abraxane ® (20 mg/kg) groups, and the toxicity of bone marrow suppression was decreased in both groups. In addition, 51.16 mg/kg Nab-PTX-PA and Abraxane ® (20 mg/kg) had similar myelosuppression toxicity, but the anti-tumor effect of the former was significantly enhanced.
As the volume ratio of organic phase to aqueous phase decreased from 1:9 to 1:22, the particle size also decreased to about 85 nm. Under the action of the emulsifier, the organic phase was dispersed into fine emulsion droplets. The smaller the concentration of the emulison droplets in the dispersed medium, the smaller the relative viscosity will be, and the more uniform the emulsion dispersion, the smaller the chance of agglomeration between emulsions will be. As a result, a relatively stable dispersion system can be formed. However, when the volume ratio decreases to a certain extent, the particle size of NPs would not change much. It is proved that the mass concentration of the carrier material affects the deposition rate of NPs in the process of ball formation. With the increase of the polymer mass concentration, the particle size increases accordingly. After the particle size decreases, it is not easy to be swallowed by the reticular endothelial system as a foreign body, the half-life is prolonged, the rate of clearance from plasma slows down, and the targeting effect is enhanced.
The drug loading and encapsulation efficiency of Nab-PTX-PA are relatively high. It is speculated that the solubility of the PTX itself is poor. After palmitic acid modification, the liposolubility is greatly improved, the LogP value is in the suitable range of 0–3, which improves the properties of the preparation and makes the drug easy to be absorbed. Nab™ technology uses the cavitation under high shear force to open the sulfhydryl or disulfide bond between albumin, and then cross-links between albumin to form new disulfide bonds, thus preparing the NPs. Because of the low temperature in the preparation process, albumin is not easy to denature. In addition, albumin contains a hydrophobic domain and has a variety of hydrophobic drug binding sites, which can better encapsulate hydrophobic drugs.
Myelosuppressive toxicity is the main side effect of Abraxane®, which can cause coagulation dysfunction and increase the risk of infection or even secondary leukemia, thus limiting its clinical use. Blood routine examination of the tumor-bearing mice showed that Nab-PTX-PA (25.58 mg/kg) could significantly reduce the toxicity of bone marrow suppression. It was speculated that when PTX was prepared into a PTX-PA prodrug, the toxicity of bone marrow suppression would be decreased according to the characteristics of the prodrug. Compared with the commercially available Abraxane®(20 mg/kg), Nab-PTX-PA (25.58 mg/kg) showed no significant difference in antitumor efficacy but had less myelosuppressive toxicity. Compared with Abraxane®(20 mg/kg), Nab-PTX-PA (51.16 mg/kg) had similar myelosuppressive toxicity but its anti-tumor effect was increased significantly. Presumably, Nab-PTX-PA at the same concentration is less toxic than Abraxane®, which provides a new research idea for the development of PTX preparations.
Nab-PTX-PA (25.58 mg/kg) showed the same anti-tumor effect as the commercial drug Abraxane ® (20 mg/kg) in the tumor-bearing mice, and the anti-tumor effect of 51.16 mg/kg Nab-PTX-PA was better than that of Abraxane ®(20 mg/kg). It is speculated that PTX-PA has high fat solubility, binds more closely to albumin and slowly forms a drug-protein complex in blood. More drugs enter tumor cells through active targeting mediated by gp60-cellar protein-SPARC. Palmitic acid is a fatty acid that can be absorbed as energy by cancer cells, so the more the drug accumulates in the tumor site, the stronger the ability to inhibit tumor growth. Albumin is an endogenous substance with no immunogenicity and toxicity. Modification of PTX with palmitic acid not only reduced the adverse effects of the drug but increased tolerance of the experimental mice to the drug. The particle size of Nab-PTX-PA is about 86 nm, and therefore the drug can penetrate and remain in the tumor site more easily through the EPR effect of the tumor, and exert a better anti-tumor effect. In addition, there may be a metabolic balance between the degradation of Nab-PTX-PA to PTX and the consumption of PTX in mice in vivo. In the tumor site, there may be a large number of metabolic esterases with different degrees of activity. After palmitic acid modification, Nab-PTX-PA is doubly targeted, and under the action of metabolic esterase, the directional release of PTX at the tumor site makes the anti-tumor effect of Nab-PTX-PA more effective. On the basis of the above research, our team will continue to study the tissue distribution, pharmacokinetics and different metabolic esterase activities of Nab-PTX-PA in vivo and try to explain the results of pharmacodynamic evaluation of Nab-PTX-PA in animals at s deeper level.