ABSTRACT
Objective: To prepare norcantharidin TPP-PEG-PCL nanomicelles and study its release in vitro, intracellular transport and promoting effect on hepatoma cell apoptosis. Methods: Thin film hydration method was used to prepare norcantharidin TPP-PEG-PCL nanomicelles, and the particle size, electric potential and microscopic electron microscopy morphological analysis were measured. At the same time, the nanomicelles were evaluated for stability, in vitro release, pharmacokinetics and critical micelle concentration. Coumarin-6 was used as a fluorescent probe to evaluate the uptake of TPP-PEG-PCL nanomicelles in liver tumor cells, lysosomal escape and mitochondrial targeting function; Under the same dosage conditions, the effect of norcantharidin TPP-PEG-PCL nanomicelles on promoting apoptosis of liver tumor cells was evaluated. Results: The cantharidin TPP-PEG-PCL nanomicelles had a particle size of (16.8 ± 0.2) nm, a Zeta potential of (14.3 ± 0.2) mV, and transmission electron microscopy images showed that nanomicelles had a regular spherical shape. The fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote the cellular uptake of drugs, escape lysosomal capture, and finally target aggregation at the mitochondrial site; Cell survival rate and Hoechst staining results showed that cantharidin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of liver tumor cells. Norcantharidin TPP-PEG-PCL nanomicelles can significantly reduce mitochondrial membrane potential, increase intracellular ROS levels, increase pro-apoptotic protein Bcl-2, and reduce resistance. The expression of apoptotic proteins Bax and these pro-apoptotic related experimental results are significantly better than those of norcantharidin PEG-PCL nanomicelles and norcantharidin, which have statistical significance. Conclusion: Norcantharidin TPP-PEG-PCL nanomicelles have good liver tumor cell mitochondrial targeting and promote tumor cell apoptosis, and it is a potentially effective drug delivery system for targeting tumor cell mitochondria.
ABSTRACT
Orthogonal experiments were used to optimize the process parameters of curcumin TPP-PEG-PCL nanomicelles; the particle size, electric potential and morphology under the electron microscope were systematically detected for the curcumin TPP-PEG-PCL nanomicelles; and the stability and in vitro release of the curcumin TPP-PEG-PCL nanomicelles were investigated. With DID fluorescent dye as the fluorescent probe, flow cytometry was used to study the uptake of nanomicelles by breast cancer cells, and laser confocal microscopy was used to study the mitochondrial targeting and lysosomal escape functions of nanomicelles. Under the same dosage conditions, the effect of curcumin TPP-PEG-PCL nanomicelles on promoting the apoptosis of breast cancer cells was evaluated. The optimal particle size of curcumin TPP-PEG-PCL nanomicelle was(17.3±0.3) nm, and the Zeta potential was(14.6±2.6) mV in orthogonal test. Under such conditions, the micelle appeared as regular spheres under the transmission electron microscope. Fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote drug uptake by tumor cells, escape from lysosomal phagocytosis, and target the mitochondria. The cell survival rate and Hoechst staining positive test results showed that curcumin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of breast cancer cells. The curcumin TPP-PEG-PCL micelles can significantly reduce the mitochondrial membrane potential of breast cancer cells, increase the release of cytochrome C, significantly increase the expression of pro-apoptotic protein Bcl-2 and reduce the expression of anti-apoptotic Bax protein. These test results were significantly better than those of curcumin PEG-PCL nanomicelles and curcumin, with statistically significant differences. The results revealed that curcumin TPP-PEG-PCL nanomicelles can well target breast cancer cell mitochondria and escape from the lysosomal capture, thereby enhancing the drug's role in promoting tumor cell apoptosis.
Subject(s)
Humans , Apoptosis , Breast Neoplasms/drug therapy , Cell Line, Tumor , Curcumin/pharmacology , Lysosomes , Micelles , Mitochondria , Phosphatidylethanolamines , Polyethylene GlycolsABSTRACT
The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endo-lysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.
ABSTRACT
The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endolysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.
ABSTRACT
Objective To prepare and evaluate As2O3 loaded polyethylene glycol-polycaprolactone-polyethyleneimine (PEG-PCL- PEI, PPP) nanoparticles (As2O3-PPP-NPs) in vitro. Methods As2O3-PPP-NPs was prepared by one-step electrostatic loading method using PPP triblock polymer as carrier. The drug loading and entrapment efficiency of the nano-drug were determined by ICP-OES. In vitro drug release property was studied by the dialysis bag method. Hemolytic toxicity of As2O3-PPP-NPs was investigated by UV spectrophotometry. Cytotoxicity of As2O3-PPP-NPs on human cervical cancer (HeLa) and human hepatocellular carcinoma cells (HepG2) was evaluated by MTT assay. Finally, ICP-OES and confocal microscopy was used to investigate the uptake efficiency and uptake mechanism of As2O3-PPP-NPs by HepG2 cells. Results The prepared nano-formulations were spherical and well-dispersed with particle size of 88.7 nm. The encapsulation efficiency and the drug loading rate were (92.75 ± 3.83)% and (4.39 ± 0.26) %, respectively. In vitro release studies showed that As2O3-PPP-NPs had the characteristics of sustained release and low pH responsive drug release, which could achieve specific drug release in the tumor environment. The loading of As2O3 neutralized the positive charge of PPP, and the hemolytic toxicity of the material was reduced. MTT assay showed that the median lethal concentrations (IC50 values) of As2O3-PPP- NPs to HeLa and HepG2 cells were 6.24 μmol/L and 5.85 μmol/L, respectively, which showed strong inhibiting effect on tumor cells. Cellular uptake studies showed that As2O3-PPP-NPs was rapidly taken up by cells due to positively charged surface and featured the lysosomal escaping ability, so the drug could be released in the cytoplasm and exert its anti-tumor effect. Conclusion As2O3-PPP-NPs exhibits significantly sustained and low pH responsive release characteristics, and has the ability to escape from lysosomes. As2O3-PPP-NPs is a potential drug delivery system against solid tumor.
ABSTRACT
Charge-reversal nanocarrier was constructed to enhance lysosomal escape and improve an-titumor effect. We synthesized the cholesterol-polyethyleneimine-hexahydrophthalic anhydride (Chol-PEI-HHPA) polymer and characterized by 1H NMR. The charge-reversal liposomes (Lipo-HHPA) were synthesized and the hematoporphyrin monomethyl ether (HMME) was loaded. pH-triggered charge conversion was determined at different pH values. The lysosomal escape and cytotoxicity of the Lipo-HHPA were evaluated in MCF-7 cells. The Lipo-HHPA was uniform with an average particle size of 102 nm. Upon the irradiation of ultrasound, burst release of HMME could be observed. The zeta potential of Lipo-HHPA changed sharply from negative (-23.5 mV) to positive (+21.2 mV) over the pH range of 7.4-4.5. In the cellular uptake experiment, the lysosomal escape of Lipo-HHPA was observed. HMME loaded Lipo-HHPA displayed obviously enhanced cytotoxicity towards MCF-7 cells. These results indicate that the charge-reversal liposomes hold a great potential in improving the cytotoxicity and antitumor effect.
ABSTRACT
OBJECTIVE: To review the progress on materials with lysosomal escape function. METHODS: Based on the original researches in recent years, the structure properties and escape mechanism of materials with lysosomal escape function were introduced in the present paper. RESULTS: The materials which can help the drug/gene to get out of the lysosomes were summarized and classified into two groups in the accordance with their escape mechanism: breaking the lysosomes by raising the osmotic pressure or destabilizing the membrane structure of lysosomes. CONCLUSION: The materials with lysosomal escape function, as a novel drug delivery tool, has promising application for the delivery of drug/gene and need to be studied deeply before the materials get widely applied.