Preparation Of Curcumin-loaded Composite Lipid Nanoparticles And Its Anti-tumor Efficacy

In this study,amorphous calcium carbonate?ACC?was used as the carrier to prepare ACC-Cur loaded with Curcumin?Cur?by gas diffusion method,and the surface was modified with phospholipid?PL?and DSPE-PEG to prepare PL/ACC-Cur.The results of particle size distribution and surface potential measurement indicate that the average particle size of ACC-Cur is about 80nm and the surface is positively charged,and the average particle size of PL/ACC-Cur is about 100nm and the surface is negatively charged.Observing the morphology of the nanoparticles using TEM revealed that both ACC-Cur and PL/ACC-Cur are core-shell nanoparticles with uniform size distribution,quasi-spherical,and isotropic structure.The results of ultraviolet spectroscopy indicate that Cur exists in the form of Ca-Cur?calcium-curcumin?complex in ACC-Cur.The results of dialysis study on drug release show that ACC-Cur has both water-sensitive and acid-sensitive drug release methods,which can effectively release the loaded Cur,while PL/ACC-Cur can be kept stable under the same conditions,pH 7.4 The cumulative drug release percentage in 24 hours of PBS?phosphate buffer solution?was only 12.98%,and the drug release in PBS at pH 5.5did not increase significantly.ACC-Cur releases a large amount of calcium(Ca²⁺)at the same time of releasing the drug,which can increase the Ca²⁺concentration in 20%FBS?fetal bovine serum?to 9.61 times in 2 hours.In contrast,PL/ACC-Cur was incubated with 20%FBS for 2 hours with almost no Ca²⁺release,and maintained good stability in PBS?pH 7.4?and mouse plasma.Taking MCF-7 as a model cell,the co-localization results in MCF-7 cells showed that the endocytosis of PL/ACC-Cur in PL/ACC-Cur was mainly regulated by lysosomes.During the maturation of lysosomes,the esterase activated by acidification of the lysate may decompose the lipid components on the surface of PL/ACC-Cur and eventually expose the core ACC-Cur.Subsequently,the ACC-Cur core decomposes and releases Cur and Ca²⁺rapidly in two ways:water-sensitive and acid-sensitive.Studies on the Ca²⁺in MCF-7 cells after incubation with different preparations showed that the intracellular Ca²⁺concentration in the PL/ACC group was much higher than that in the PL/CCC group,suggesting that ACC can release Ca²⁺more quickly than CCC.At the same time,the intracellular Ca²⁺concentration in the PL/ACC-Cur and PL/CCC-Cur groups were higher than that in the PL/ACC and PL/CCC groups,respectively,and both were higher than in the free Cur group.Among them,the highest concentration of Ca²⁺in PL/ACC-Cur cells indicates that Cur is beneficial to prevent the transport of cytoplasmic Ca²⁺,which provides a prerequisite for PL/ACC-Cur-induced intracellular Ca²⁺overload.The co-localization of Ca²⁺and mitochondria in cells at different incubation time points indicates that after PL/ACC-Cur degradation,a large amount of Ca²⁺will flood into mitochondria.Mitochondrial swelling test showed that compared with blank cells,the PL/ACC group did not show any phenomenon of mitochondrial swelling,suggesting that PL/ACC is not toxic to cells,which may be related to the efficient Ca²⁺transport ability of tumor cells.This function It can maintain the Ca²⁺balance in tumor cells.Both free Cur and PL/CCC-Cur cause a certain degree of mitochondrial swelling.In contrast,PL/ACC-Cur can cause strong mitochondrial swelling,causing severe damage to the mitochondria of MCF-7 cells.The expression levels of a series of apoptosis-related protein signaling molecules such as cytochrome C,activated?Cleaved Caspases-3?and unactivated Caspases-3 and Bcl-2 were determined by Western Blot experiments.Inside,it leads to the swelling of mitochondria,the destruction of the structure,the release of cytochrome C,the activation of Capspase-3 and the downregulation of Bcl-2,which eventually leads to apoptosis.Quantitative determination of the time-dependent ATP in MCF-7 after treatment with different preparations showed that both free Cur and PL/CCC-Cur caused a certain degree of ATP level decline.In contrast,the ATP level in the PL/ACC-Cur group decreased sharply,only 19.5%of blank cells after 12h,suggesting that the severe damage caused by PL/ACC-Cur to the mitochondria of MCF-7 cells would cut off the energy of the cells source.The results of in vitro cytotoxicity experiments show that the cell survival rate of the two cells after treatment with PL/ACC,MCF-7 and L02,exceeds 90%at all concentrations,and has good biological safety.As a chemotherapeutic drug with high tumor cell specificity,under the same conditions,Cur is much more toxic to MCF-7cells than L02 cells.It is worth mentioning that the PL/ACC-Cur cytotoxicity to MCF-7 cells at low Cur concentration?1-2.5?g/mL?is comparable to that of free Cur.When the concentration of Cur administered exceeds 5?g/mL,the cytotoxicity of PL/ACC-Cur,especially to MCF-7 cells,increased significantly and was higher than that of free Cur,suggesting that Ca²⁺concentration is crucial to the effect of PL/ACC-Cur.In addition,under the same conditions,the cytotoxicity of PL/ACC-Cur on MCF-7 cells was much higher than that of PL/CCC-Cur.The effects of different preparations on the proliferation of MCF-7 cell spheres were investigated.The results show that free Cur and PL/CCC-Cur can only delay the growth of cell sphere volume.However,under the same administration conditions,PL/ACC-Cur can induce apoptosis and effectively kill tumor cells to achieve negative growth of cell sphere volume.It is suggested that PL/ACC-Cur around 100nm can achieve long circulation characteristics in vivo,avoid premature recognition and excretion by RES,and achieve better tumor targeting effect through EPR effect.An MCF-7 tumor-bearing nude mouse animal model was established.In the in vivo pharmacodynamic evaluation,the PL/ACC-Cur model animal drug efficacy was comprehensively evaluated through two indicators of tumor growth curve and life cycle.The results showed that the nude mice in the PL/ACC-Cur group had the slowest tumor volume growth,the longest survival period,and the best anti-tumor effect.The body weight analysis and the pathological analysis results of the HE sections of the main organs showed that all Cur preparation groups had no obvious damage to the main organs,suggesting that Cur,as a natural chemotherapy drug,has lower toxicity and side effects.