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Back to Journal »International Journal of Nanomedicine» Volume 16

Anti-tumor effect of hyperoside with charge reversal and mitochondrial targeting liposomes

Authors: Feng Yan, Qin Gang, Chang Sheng, Jing Ze, Zhang Yan, Wang Yan

Published on April 28, 2021, the 2021 volume: 16 pages 3073-3089

DOI https://doi.org/10.2147/IJN.S297716

Single anonymous peer review

Editor who approved for publication: Dr. Farooq A. Shiekh

Yufei Feng,1 Guozhao Qin,1 Shuyuan Chang,1 Zhongxu Jing,2 Yanyan Zhang,1 Yanhong Wang1 1 Heilongjiang University of Traditional Chinese Medicine, Ministry of Education Key Laboratory of Chinese Medicine, Harbin, Heilongjiang; 2 Heilongjiang Provincial Administration of Traditional Chinese Medicine, Harbin, Heilongjiang, People’s Republic of China Corresponding author: Yanhong Wang Tel +86451-87266893 Email [email protected] Introduction: Hyperoside (HYP), a flavonol glycoside compound, has been shown to significantly inhibit the proliferation of malignant tumors. Mitochondria are not only the “energy factory” of the cell, but also the “suicide weapon arsenal” of the cell. Targeted delivery of cytotoxic drugs to the mitochondria of tumor cells and tumor vascular cells is a promising strategy to improve the efficacy of chemotherapy. OBJECTIVE: We report a new bifunctional liposome system with extracellular charge reversal and mitochondrial targeting characteristics to enhance drug accumulation in mitochondria and induce apoptosis of cancer cells. Method: Using L-lysine as the linker, connect 2,3-dimethylmaleic anhydride (DMA) and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine (DSPE) to obtain a new Compound DSPE-Lys-DMA (DLD). Then, DLD is mixed with other commercially available lipids to form charge reversal and mitochondrial targeting liposomes (DLD-Lip). The size, shape, zeta potential, serum stability and protein adsorption of DLD-Lip (HYP/DLD-Lip) loaded with HYP were measured. The release profile of HYP/DLD-Lip, cell uptake, toxicity in vitro and in vivo, and anticancer activity were studied. Results: The results showed that the average diameter of liposomes was less than 200 nm. The zeta potential of liposomes is negative at pH 7.4. However, with the cleavage of the DMA amide, the zeta potential becomes positive at a weakly acidic pH. The charge reversal of HYP/DLD-Lip promotes cell internalization and mitochondrial accumulation, thereby enhancing the anti-tumor effect. The strongest tumor growth inhibition (TGI 88.79%) was observed in the CBRH-7919 tumor xenograft BALB/C mice treated with DLD/HYP-Lips, and there was no systemic toxicity. Conclusion: Charge reversal and mitochondrial targeting liposomes represent a promising anti-cancer drug delivery system that can enhance the effect of anti-cancer treatment. Keywords: mitochondrial targeting, liposomes, charge reversal, anti-tumor, hyperoside

Traditional Chinese medicine has a long history of treating tumors, among which paclitaxel and vincristine have significant anti-tumor effects and are widely used clinically. 1-3 In recent years, the flavonol glycoside compound HYP extracted from Hypericum perforatum and Phyllanthus emblica has been widely used. It has been shown to significantly inhibit the proliferation of malignant tumors. 4,5 The anti-tumor mechanism of HYP is mainly through the regulation of calcium-related mitochondrial pathways. 6 Mitochondrial calcium ion overload can induce the release of cytochrome C and apoptosis-inducing factors from the mitochondria into the cytoplasm, and eventually lead to apoptosis. 7,8 However, like most chemotherapeutics, some shortcomings of HYP, such as low solubility and lack of mitochondrial targeting, limit its further application. 9,10 Therefore, it is urgent to design and prepare a safe and effective drug delivery system to improve the anti-tumor effect of HYP.

Designing tumor microenvironment stimulating drug delivery systems based on the unique microenvironment of tumor tissues, such as stimulus-responsive nanoparticles, microcapsules, polymer-drug conjugates and prodrugs, is expected to improve the effect of anticancer drugs. 11-13 Liposomes, which are often used as carriers of anti-tumor drugs, are vesicles with a cell membrane structure composed of phospholipids and cholesterol. Due to its good biocompatibility and degradability, the carrier can be enhanced by penetration and retention (EPR) in the tumor. This has been extensively studied due to the accumulation of tumor sites and the ability to load hydrophilic and hydrophobic drugs. 14,15 However, as the blood circulates, liposomes are easily captured and eliminated by the reticuloendothelial system and the mononuclear macrophage system. Polyethylene glycol (PEG) modification can increase the circulation time of liposomes in the blood, 16,17 but its presence will affect the efficiency of drug entry into cells. Since the metabolism of tumor tissues mainly relies on anaerobic glycolysis, the metabolites, lactic acid and CO2 produced by the respiration of tumor tissues lead to acidification of the tumor microenvironment. Tumor tissues are characterized by low pH, which means that the pH value of the tumor cell interstitium (6.8) is lower than that of normal tissues (7.4); the pH value of lysosomes is 4.5.18, which allows preparations with a controllable pH response Of liposomes to increase the possibility of anti-cancer activity. Therefore, more research has focused on the functionalization of liposomes with long-cycle characteristics of PEG de-shielding mechanism, that is, through the degradable pH-sensitive bonds between PEG and lipids, such as hydrazone and hydrazine bonds. 19-25 In an acidic environment, acid-sensitive chemical bonds will remove PEG on the surface of liposomes and enhance cell uptake. 26 Liposomes can also be decorated with carboxylic acid amide 27 or maleic anhydride amide 28, 29, which can be used to achieve charge reversal. In the blood, negatively charged liposomes can avoid non-specific protein adsorption and prolong circulation time. However, when these liposomes are exposed to the weakly acidic environment of tumor tissues, the amide bond will be destroyed, thereby generating a positive charge on the liposome surface, thereby significantly increasing the liposome's cellular internalization30.

Based on the characteristics of tumor tissue microenvironment and the effect of HYP on mitochondria, this study constructed a new type of liposome drug delivery system. A pH-responsive DSPE-Lys-DMA (DLD) lipid was designed. L-Lysine is used as a linker to connect 2,3-dimethylmaleic anhydride (DMA) and 1.2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to produce a new compound DLD. Mix pH-responsive DLD with other commercially available lipids to form charge reversal, mitochondrial targeted liposomes (DLD-Lip). At pH 7.4, liposomes are negatively charged due to the carboxyl group in 2,3-dimethylmaleic anhydride. When DLD-Lip accumulates in tumor tissue, the weak acidity in the microenvironment destroys the dimethylmaleamide bond, and the positively charged amino group is exposed. The surface charge of DLD-Lip changes from negative to positive. Tumor cell membranes, thereby increasing the uptake of tumor cells. Then, the electrostatic interaction between the highly positively charged liposome and the mitochondrial membrane (130~150 mV, internal negative charge) causes it to accumulate in the mitochondria, achieve mitochondrial targeting, and finally trigger HYP to exert its therapeutic effect (Scheme 1). Scheme 1 Diagram showing the pH response of dual-functional liposomes (DLD-Lip) to tumor microenvironment and mitochondrial targeted anticancer drug delivery. a) The pH response of DLD in the weakly acidic tumor microenvironment for surface charge conversion. b) The positive charge of liposomes improves cellular uptake. c) The liposomes are internalized into tumor cells. d) Combine with mitochondria. e) HYP plays a therapeutic role in mitochondria. f) Mitochondrial apoptosis.

Scheme 1 Diagram showing the pH response of dual-functional liposomes (DLD-Lip) to tumor microenvironment and mitochondrial targeted anticancer drug delivery. a) The pH response of DLD in the weakly acidic tumor microenvironment for surface charge conversion. b) The positive charge of liposomes improves cellular uptake. c) The liposomes are internalized into tumor cells. d) Combine with mitochondria. e) HYP plays a therapeutic role in mitochondria. f) Mitochondrial apoptosis.

Hyperin reference substance (batch number: 111521-201708) was purchased from China Institute for the Control of Pharmaceutical and Biological Products. 1.2-Distearoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (DSPE-mPEG2000) was purchased from Shanghai Fansheng Biotechnology Co., Ltd. Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Rat liver cancer cell line CBRH-7919 cells were purchased from Shanghai Lianmai Bioengineering Co., Ltd. Mitochondrial dissection, Caspase 3 activity detection kit, Caspase 9 activity detection kit, Annexin V- FITC cell apoptosis detection kit was purchased from Biyuntian Biotechnology Co., Ltd. Female BALB/c nude mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China, license number: SCXK (Beijing) 2016-0006, initial weight 18) – 22 g) and placed in the animal experiment center. All related operations of experimental animals are approved and implemented in accordance with the relevant requirements of the Animal Experiment Management Committee of Heilongjiang University of Traditional Chinese Medicine.

Under a nitrogen atmosphere, dissolve DSPE (1.50 g, 2.00 mmol) in anhydrous chloroform (30 mL). DIPEA (2.00 mL, 12.00 mmol) was added to the solution under stirring at 0°C. Add Boc-L-Lys(Boc)-OH (0.83 g, 2.40 mmol), HOBT (0.32 g, 2.40 mmol) and HBTU (0.91 g, 2.40 mmol) dissolved in dry DMF (5 mL) to the reaction The flask contains DSPE. The solution was stirred in an ice bath under nitrogen for 30 minutes and then at room temperature for another 24 hours. The solvent was removed using rotary evaporation and the mixture was purified by column chromatography (silica, CH2Cl2:MeOH=10:1). The product DSPE-(Boc)-L-Lys-Boc was vacuum dried.

Dissolve the received DSPE-(Boc)-L-Lys-Boc in anhydrous dichloromethane (DCM)/TFA (1:1, 10 mL). The solution was stirred under nitrogen for 4 hours. The solvent was removed by rotary evaporation, and then the sample was vacuum dried for 1 hour. The product precipitated in anhydrous ether. The white precipitate was washed 3 times with anhydrous ether and collected by centrifugation. After vacuum drying, DSPE-Lys was obtained with a yield of 80.23%. The 1H NMR spectrum of DSPE Lys is shown in the supporting information.

Under the protection of nitrogen, dissolve DSPE-Lys (1.00 g, 1.14 mmol) in 20 mL of anhydrous dichloromethane. In an ice bath, triethanolamine (TEA, 0.58 g, 5.71 mmol) and DMA (0.57 g, 4.56 mmol) dissolved in dichloromethane were injected into the reaction system, and the reaction system was stirred at room temperature for 24 hours. After the reaction, the organic solvent was removed, and the crude product was recrystallized in an acetone solution at 4°C. Finally, the resulting precipitate DSPE-Lys-DMA was washed 3 times with ether, and then dried under vacuum. The yield was 30%. H-nuclear magnetic resonance (1H-NMR) and electrospray ionization mass spectrometry (ESI-MS) were used to confirm the structure of DSPE-Lys. The 1H NMR spectrum of DSPE-Lys-DMA is shown in the supporting information.

The membrane hydration method was used to prepare HYP-loaded liposomes. Use chloroform/methanol as a mixed solvent (2:1, v /v). Use a rotary evaporator to remove the organic solvent, form a uniform film on the bottom of the bottle, and vacuum dry overnight. The film dried overnight was hydrated in phosphate buffered saline (PBS, pH 7.4) or ultrapure water to a phospholipid concentration of 1.6 mg/mL. The liposomes were sonicated in a 37°C water bath for 10 minutes, and then probed for a further 100 seconds at 40 W to prepare DLD/HYP-Lip. Add DLD/HYP-Lip (0.5 mL) to 2.0 mL ultrapure water, place it in an ultrafiltration centrifuge tube, and centrifuge at 4000 rpm for 20 min. Then, the lower layer of the solution was removed, and the concentration of HYP was measured by high performance liquid chromatography (HPLC). Calculate the encapsulation efficiency and drug loading according to the following formula:

W: HYP content in liposomes after centrifugation

W0: HYP content in liposomes

W medicine: the weight of HYP

W total: the weight of HYP, cholesterol and phospholipids

A dynamic light scattering (DLS) analyzer was used to measure the average particle size of the liposomes. For the sample, dilute 100 μL of liposomes (phospholipid concentration 5 mg/mL) in 1 mL of buffer solution.

DLD/HYP-Lip (200 µL) is diluted to a certain multiple, added with a special copper mesh, and dyed with phosphotungstic acid. The sample was then air-dried, and then the shape of the particles was observed under a transmission electron microscope.

In order to study the potential changes of DLD/-Lip in different pH buffer solutions, 1 mL of SPC-Lip and DLD/-Lip were added to 15 mL of PBS solution (pH 7.4, pH 6.8) and acetic acid buffer solution (at pH 5.5 and pH 4.5), 37°C, 0, 2, 4, 6, 8 and 12 hours. Take 1.5 mL sample for potential measurement.

In order to study the interaction between the drug and other excipients during the preparation of liposomes, the crystal properties of HYP and other excipients were analyzed by differential scanning calorimetry (DSC). Weigh an appropriate amount of sample and place it in an aluminum crucible, and seal the crucible lid. The blank crucible is used as a reference. The temperature range is 40°C to 300°C, the heating rate is 20°C/min, and the flow rate of the shielding gas (high-purity nitrogen) is 60 mL/min. Analyze SPC, cholesterol, DSPE-PEG, DLD, HYP, DLD/HYP-Lip, DLD/HYP-Lip physical mixed samples, and record the differential scanning calorimeter characteristic curve of each sample.

The stability of liposomes in serum was evaluated by measuring the particle size changes of SPC-Lip and DLD-Lip in fetal bovine serum. Liposomes with a phospholipid concentration of 5 mg/mL were mixed with an equal volume of fetal bovine serum and incubated at 37°C. At 1, 4, 8 and 24 hours, 100 μL of sample was taken for particle size determination. The liposome particle size at 0 h is the particle size measured in a pH 7.4 PBS solution.

Bovine serum albumin (BSA) was used as a model protein to test the protein adsorption capacity of liposomes. The liposomes were placed in a pH 7.4 or 6.8 phosphate buffer solution (phospholipid concentration of 0.15 mg/mL and protein concentration of 0.25 mg/mL) containing BSA and incubated at 3°C ​​for 2 hours or 4 hours, respectively. Centrifuge 300 μl of the solution at 13,000 × g for 15 minutes. The adsorbed protein aggregates settle at the bottom, and the supernatant contains unadsorbed protein. Using BSA as the standard solution, the protein content in the supernatant was determined with a bicinchoninic acid (BCA) protein kit. Then, add 25 μL of sample to a 96-well plate and add 200 μL of BCA reaction solution. The microplate reader measures the absorbance at 570 nm.

The in vitro release of DLD/HYP-Lip in the release medium is determined by dialysis. Specifically, 0.1 mL of DLD/HYP-Lip and free HYP solution (HYP content 0.5 mg/mL) were added to the pretreated dialysis bag (molecular weight cut-off 6000-8000 Da), and placed in 50 mL release medium. The samples were then centrifuged at 50 rpm at 37°C. The release medium is 0.1% Tween 80 in PBS solution (pH 7.4, 6.8) and 0.1% Tween 80 acetate buffer solution (pH 5.5, 4.5). At 0.5, 1, 2, 4, 8, 12, and 24 hours, collect 0.5 mL of release medium and add an equal volume of fresh release medium to the drug release medium. Determine the content of HYP in the sample, and calculate the cumulative release at each time point by the following formula. Then draw the release curve.

Among them, Er represents the cumulative release of HYP, Ve represents the displacement volume of the release medium, Ci represents the concentration of the drug released during the i-th sample replacement, V0 represents the volume of the release medium, and Cn represents the release medium in the nth sample. The drug concentration Mdrug represents the HYP content in the liposome.

CBRH-7919 cells are derived from rat liver cancer cells. Compared with primary liver cancer cells, they are passaged, making it easier to culture in vitro. CBRH-7919 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% diabody.

In order to observe the in vitro uptake of DLD/HYP-Lip by CBRH-7919 cells, the cellular uptake of DLD/HYP-Lip was detected by flow cytometry. CBRH-7919 cells were seeded in parallel in a 6-well plate, 1×105 per well. Incubate the plate in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhere to the wall, aspirate the original culture medium and wash twice with PBS. After washing, discard PBS, and add DLD/HYP-Lip solution (pH 7.4 and pH 6.8) at a concentration of 0.5 μg/mL. A fresh medium without any drugs was used as a control group. Incubate the sample for 4 hours. After incubation, aspirate the original culture solution, wash with cold 4°C PBS 3 times, add 500 μL trypsin digestion solution, and then add 2 mL complete medium to terminate the digestion. All adherent cells were suspended in complete medium and centrifuged (1000 rpm, 5 minutes). Discard the supernatant and resuspend the cells in 1 mL of 4°C PBS. The sample was then centrifuged (1000 rpm, 5 min) twice, and finally the cells were dispersed in 300 μL of 4°C PBS. These cells are then used in flow cytometry to analyze the fluorescence intensity.

Free HYP, SPC/HYP-Lip, pH-sensitive DLD/HYP-Lip and blank DLD-Lip have in vitro cytotoxicity to CBRH-7919 cells at pH 7.4 and pH 6.8. Use 3-(4,5-dimethyl-thiazole) -2-yl)-2,5-diphenyl-tetrazolium bromide (measured by MTT). CBRH-7919 cells were seeded in a 96-well plate in parallel, with 5×104 cells per well. After 24 hours of incubation, the cells were exposed to a fresh medium adjusted to pH 7.4 or 6.8, which contained blank or HYP-loaded liposomes. After 48 hours of incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the samples were incubated for another 4 hours. Measure the absorbance (OD490) at the wavelength of 490 nm of the microplate reader.

The mitochondrial localization of SPC/HYP-Lips and DLD/HYP-Lips in CBRH-7919 cells was observed under a laser confocal scanning microscope. Inoculate CBRH-7919 cells (1 × 104 cells in each glass bottom culture dish) and incubate at 37°C under 5% CO2 for 24 hours. After 24 hours of incubation, the cells were treated with SPC/HYP-Lips or DLD/HYP-Lips and cultured under the same conditions for another 12 hours (final HYP concentration: 0.5 μg/mL). After incubation, the medium was removed, the cells were washed 3 times with cold PBS, and then stained with Mitotracker Green FM (75 nM) at 37°C and 5% CO2 for 30 minutes. The stained cells were washed 3 times with PBS to remove free dye and observed by CLSM.

CBRH-7919 cells were seeded in parallel in a six-well plate with 1×105 cells per well. Incubate the plate in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhere to the wall, aspirate the original culture medium, add 2 mL, and wash the sample twice with PBS. Then SPC/HYP-Lip and DLD/HYP-Lip solutions with a concentration of 0.5 μg/mL were added; fresh medium without any additives was used as the control group. The samples are incubated in a cell incubator for 24 hours. After incubation, aspirate the original culture medium, wash twice with cold 4°C PBS, and then extract mitochondria using a mitochondrial dissection kit. The cells were re-dispersed in the mitochondrial stripping buffer, homogenized 15 times in a homogenizer, the resulting suspension was centrifuged (13,000 rpm, 10 min), the supernatant was collected, and centrifuged again (11,000 rpm, 10 min). For mitochondrial separation, the required precipitate is mitochondria. After aspirating the supernatant, the mitochondria are re-dispersed in 300 μL PBS. Use flow cytometry to measure the HYP content in mitochondria.

CBRH-7919 cells were seeded in parallel in a 6-well plate, 1×105 per well. Incubate the plate in a 37°C, 5% CO2 cell incubator for 24 hours. After the cells adhere to the wall, aspirate and add the original culture medium. Then the cells were washed twice with 2 mL PBS, SPC/HYP-Lip, DLD/HYP-Lip, and HYP solution at a concentration of 20 μg/mL was added. The fresh medium without any drugs was used as the control group. The cells were then incubated for 12 hours. In accordance with the method in the Caspase 3 and Caspase 9 activity detection kit, aspirate and use the cell culture medium. The adherent cells were digested with 500 μL trypsin and collected in the spare cell culture medium. Then centrifuge the cells (1000 rpm, 5 min), discard the supernatant, and add 2 mL PBS to wash the cells. Then centrifuge the cells (1000 rpm, 5 min), discard the supernatant, add 100 μL of lysate, and transfer the sample to a 600 μL centrifuge tube. In an ice bath, the samples were lysed for 15 minutes and then centrifuged at 4°C (16,000 rpm, 15 minutes). Transfer the supernatant to a pre-cooled centrifuge tube in an ice bath. Then, take a 5 μL sample to determine the protein concentration according to the Bradford method. Add appropriate samples to Caspase 3 and Caspase 9 substrates, and incubate overnight at 37°C in the dark. The activity of Caspase 3 and Caspase 9 was calculated by ELISA at 405 nm.

The Annexin V-FITC Apoptosis Detection Kit was used to measure the apoptosis of CBRH-7919 cells and analyzed by flow cytometry. The cells were seeded in parallel in a 6-well plate, 1×105 per well, and cultured for 24 h. Add HYP, SPC/HYP-Lip and DLD/HYP-Lip to each well, add 20 μg/mL HYP and incubate for 12 hours. After incubation, aspirate the culture solution and wash 3 times with 4°C PBS; then add 500 μL trypsin digestion solution, add 2 mL complete medium, stop the digestion and centrifuge (1000 rpm, 5 min). Discard the supernatant, resuspend the cells in PBS and centrifuge (1000 rpm, 5 minutes). According to the Annexin V-FITC apoptosis detection kit, the cells were resuspended in 195 μL Annexin V-FITC binding solution, 5 μL Annexin V-FITC and 10 μL PI staining solution. The samples are incubated at room temperature in the dark. After 20 minutes, the assay was quantified by flow cytometry.

Animal experiments were carried out in accordance with the Laboratory Animal Management Committee of Heilongjiang University of Traditional Chinese Medicine (No. 2017042501). About 5×106 CBRH-7919 cells were suspended in PBS (50 mL), and BALB/c nude mice were subcutaneously injected into the left back of anesthetized nude mice. Calculate the tumor volume (V) by measuring the length (L) and width (W), and calculate it as V=L×W2/2s. It is administered when the tumor size is 50-100 mm3.

The mice were randomly divided into four groups (n=5). Normal saline, HYP (6.0 mg/kg), SPC/HYP-Lip (6.0 mg/kg, calculated according to HYP content) or DLD/HYP-Lip (6.0 mg/kg, calculated according to HYP content) are administered via tail vein injection .31 Different formulations were injected 12, 14, 16, 18 and 20 days after CBRH-7919 cell injection. The mice were weighed and tumors were measured every 2 days. The mice were sacrificed on the 25th day after tumor inoculation, the tumors were removed, then embedded in paraffin and cut with a microtome (5 mm thick), and then stained with hematoxylin and eosin (HE). For the quantification of proliferating cells, Ki67 was evaluated with anti-Ki67 [SP6] antibody (Abcam, UK). The Ki67 index is calculated as the ratio of proliferating cells to total cells in each field, using five random fields. For the quantification of apoptotic cells, the tdt-mediated dutp notch end labeling (TUNEL) assay is used together with the in situ cell death detection kit-POD (Roche Group, Switzerland). The apoptotic index is calculated as the ratio of apoptotic cells to total cells in each field, using five random fields.

Healthy male BALB/c were randomly divided into four groups (n=5), normal saline, HYP (6.0mg/kg), SPC/HYP-Lip (6.0mg/kg, calculated according to HYP content) or DLD/HYP- Lip ( 6.0 mg/kg, calculated by HYP content) Administer via tail vein injection. Different preparations were injected, and mice were weighed every 2 days. Then, evaluate the animal behavior.

Seven days after the administration, the mice were allowed to recover and weighed daily. After execution, the liver, heart, spleen, lung, and kidney were taken, washed with PBS, fixed with 4% formaldehyde, embedded in paraffin, and sectioned (5mm thick) for HE staining.

HYP was measured by HPLC using Shimadzu HPLC system consisting of LC-20AT pump and SPD-20A diode array detector (Shimadzu Technologies). A Diamonsil C-18 column (250 mm × 4.6 mm, 5 μm; DIKMA Technologies) and a detector wavelength of 360 nm were used. A mixed solvent of acetonitrile-0.1% phosphoric acid solution (22:78, v/v) was used as the eluent, and the flow rate was 1.0 mL/min.

All data are expressed as mean ± SD. Student's t-test or one-way analysis of variance (ANOVA) is used to determine statistical significance. The statistical significance is set to *p<0.05, **p<0.01 indicates a highly significant difference.

The synthetic route of DSPE-Lys-DMA can be found in Scheme S1 of Supporting Information. The carboxyl group of Boc-L-Lys(Boc)-OH is combined with the primary amino group of DSPE and deprotected with trifluoroacetic acid. Use an amidation reaction to attach DMA to the amino group of DSPE-Lys. DSPE-Lys-DMA uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 1H NMR in the supporting information for characterization.

See Table S1 for the size, polymer dispersion index (PDI), encapsulation efficiency and drug loading of blank and HYP-loaded liposomes (SPC-Lip, DLD-Lip, SPC/HYP-Lip and DLD/HYP-Lip) support information. The average diameter of the two blank liposomes was similar, about 110 nm, while the average diameter of the two HYP drug-loaded liposomes was about 130 nm. Because the drug is encapsulated in the lipid bilayer of liposomes, the particle size of HYP liposomes is larger than that of blank liposomes. 24,32 DLD-Lips meter showed higher encapsulation efficiency and drug loading. The liposome encapsulation rate obtained by long-circulation, pH-sensitive hyperoside liposome reconstruction is (94.36±1.52)%, which is greater than 90%, indicating the best formulation of liposomes and HYP encapsulation ability powerful. 33. The loading capacity of liposomes obtained after drug reconstitution was 8.5%, indicating that liposomes prepared by optimized formulations have strong drug loading capacity for HYP. 34,35 PDI is (0.164±0.062), indicating that the liposome has a very large particle size. uniform. Transmission electron microscope (TEM) is used for morphological analysis of DLD-Lips loaded with DLD/HYP-Lips. The liposomes are round and spherical, as shown in Figure 1A. Figure 1 The physical and chemical properties of liposome preparations. TEM image of DLD/HYP-Lips (scale bar = 200 nm) (A). The particle size changes of SPC-Lip and DLD-Lip in fetal calf serum (mean ± SD, n=3) (B). Changes in Zeta potential of DLD/HYP-Lips at different pH values ​​(C). DLD/HYP-Lip, DLD, HYP, cholesterol, SPC, DSPE-PEG and DLD/HYP-Lip constitute the physical mixed sample DSC curve (D). The cumulative release of DLD/HYP-Lip in the release medium (mean ± SD, n=3) (E). After 2 and 4 hours of incubation at 37°C, pH 7.4 or 6.8 (mean ± SD, n = 3) (F), the adsorption of bovine serum albumin (BSA) on liposomes. ** p <0.01.

Figure 1 The physical and chemical properties of liposome preparations. TEM image of DLD/HYP-Lips (scale bar = 200 nm) (A). The particle size changes of SPC-Lip and DLD-Lip in fetal calf serum (mean ± SD, n=3) (B). Changes in Zeta potential of DLD/HYP-Lips at different pH values ​​(C). DLD/HYP-Lip, DLD, HYP, cholesterol, SPC, DSPE-PEG and DLD/HYP-Lip constitute the physical mixed sample DSC curve (D). The cumulative release of DLD/HYP-Lip in the release medium (mean ± SD, n=3) (E). After 2 and 4 hours of incubation at 37°C, pH 7.4 or 6.8 (mean ± SD, n = 3) (F), the adsorption of bovine serum albumin (BSA) on liposomes. ** p <0.01.

Check the stability of SPC-Lip and DLD-Lip in the serum using environmental simulation physiological conditions containing 50% fetal bovine serum. The result is shown in Figure 1B. In the pH 7.4 serum, the average particle size and distribution of DLD-Lips hardly changed after 24 hours (PDI = 0.19±0.03). These results indicate that DLD-Lips did not aggregate in the serum solution, and the average particle size and distribution of sulfate did not change significantly. 33

In order to prove that DLD-Lips change surface characteristics in response to tumor extracellular and intracellular environment, when DLD-Lips are dispersed in buffer solutions with different pH values ​​(pH 7.4, 6.8, 5.5, and 4.5), their zeta potentials are tested. These pH values ​​mimic physiological pH (pH 7.4), tumor microenvironment (pH 6.8), endosomes and lysosomes (pH 5.5), and advanced lysosomes (pH 4.5). As shown in Figure 1C, when the pH value is 7.4, the zeta potential of DLD-Lips is negative. When the pH value drops to 6.8, the surface charge of DLD-Lips immediately increases to neutral and stabilizes to 5 mV within 1 h When the pH value is 5.5 and 4.5, DLD-Lips maintains a positively charged surface, and the zeta potential is ~10 mV and 20 mV, respectively. In contrast, traditional liposomes (SPC-Lips) do not perform charge conversion. This indicates that DLD-Lip is negatively charged (pH 7.4) in the circulation and interacts less with negatively charged plasma proteins, resulting in a high degree of resistance to non-specific protein absorption and enhanced stability in the blood. 36-38 Since tumor cell membranes are usually negatively charged, when DLD-Lips accumulate at the tumor site (pH 6.8) through the EPR effect, DLD-Lips charge conversion should enhance cell absorption. 25,39 The charge recovery is due to the unstable cleavage of the amide formed between the acid-amine and DMA. It can be concluded that DLD-liposomes are negatively charged (pH 7.4) when circulating in the bloodstream, and the charge will be reversed to positive in the weakly acidic tumor microenvironment and endosomal compartment. Compared with other acid-sensitive chemical bonds (such as hydrazine-modified liposomes that cleave at pH 5.0), the amide formed between amine and DMA has an effect on the weakly acidic tumor microenvironment (pH 6.8), showing an ideal pH response. 19,40

Figure 1D shows that the characteristic melting point of SPC is about 230°C, the characteristic melting point of cholesterol is about 150°C, the characteristic melting point of DSPE-PEG is about 54°C, and the characteristic melting point of DLD is about 50°C. The peak melting point of HYP is about 255°C. The melting points of the above compounds are consistent with those reported in the literature. 41-44 The above five characteristic peaks appear in DLD/HYP-Lip physical mixed samples. The characteristic melting point peak of DLD/HYP-Lip powder appears at about 210°C, which is different from the curve of a physical mixture. Displacement occurred, and it is assumed that the DLD/HYP-Lip powder is not purely mixed physically. The research results also show that the preparation of DLD/HYP-Lip is successful.

The release characteristics of drug-loaded HYP liposomes in release media with different pH values ​​were determined. The results shown in Figure 1E show that the cumulative release of HYP in DLD/HYP-Lips at all pH values ​​after 24h is about 20%, indicating that the weakly acidic release medium only promotes charge reversal, and does not destroy the liposome structure to promote drug release . The HYP released from DLD/HYP-Lips after 48 hours is approximately 50% at all pH values, and the HYP released from DLD/HYP-Lips at all pH values ​​is approximately 60% after 72 hours. Therefore, DLD-Lips not only has a charge reversal function, but also maintains the sustained release of HYP. This slow release can prevent the sudden release of the carrier during the delivery process in the body and increase the accumulation of the drug in the tumor tissue. 45,46

The surface properties of liposomes have a greater impact on the amount of protein adsorption, and the interaction between them indirectly reflects their circulation time in the blood. If the adsorption of non-specific proteins on the liposome surface is reduced, its circulation time in the blood will be prolonged. 47,48 Therefore, we used the BCA protein experiment to determine the amount of protein adsorbed on the liposome surface. BSA is used as a model protein to mimic plasma protein. The isoelectric point of BSA is 4.5-5.0,49 under the conditions of pH 7.4 and 6.8, PI value is less than the isoelectric point (PI), and the surface of BSA is negatively charged, pHs will not affect the charge on the surface of BSA, so the cell adhesion In the attached experiments and blood compatibility experiments, BSA is usually used as a model protein to test the protein adsorption capacity of liposomes under the conditions of pH 7.4 and 6.8. As shown in Figure 1F, at a physiological pH of 7.4, after protein and liposomes are incubated for 2 hours, the amount of protein adsorbed on the surface of SPC-Lip is (60.97±2.86), DLD-Lip is (30.10±2.14), DLD-Lip Significantly smaller than the SPC-Lip surface. When the incubation time was extended to 4 h, the amount of protein adsorption on the surface of SPC-Lip was (59.84±3.59), DLD-Lip was (34.87±2.91), and there was no significant increase in SPC-Lip and DLD-lip compared to 2 h , The change in the test group was the same as that of 2 h. However, when liposomes and proteins are exposed to pH 6.8 for 2 hours, the amount of protein adsorbed on the surface of SPC-Lip is (54.89±2.94), DLD-Lip is (69.98±5.07), and the interaction between DLD-Lip and protein is strong , About 70% of the protein is absorbed by the liposome surface. When the incubation time is extended to 4 h, the amount of protein adsorption on the surface of SPC-Lip is (57.60±3.70), DLD-Lip is (79.04±6.10), and the interaction between DLD-Lip and protein is also very strong, about 80% The protein is absorbed by the liposome surface. The result proves that the change of DLD charge leads to the change of protein adsorption effect. These results are consistent with the charge reversal phenomenon of DLD-Lip at pH 6.8.

In order to observe the in vitro uptake of DLD/HYP-Lip by CBRH-7919 cells, the autofluorescence of HYP was used as a fluorescent label, and the cellular uptake of DLD/HYP-Lip was studied by flow cytometry. The fluorescence intensity is quantified in the graph. Different fluorescence intensities are used to indicate liposome uptake. The cell uptake analysis results of DLD/HYP-Lip are shown in Figure 2A and B. For ordinary liposomes, the pH does not change the uptake. In contrast, DLD/HYP-Lips are greatly affected by the pH of the environment. The average fluorescence intensity of SPC/HYP-Lips is similar in CBRH-7919 cells. When the pH is 7.4, CBRH-7919 cells absorb very few liposomes, and the average fluorescence intensity is (9.81±0.71). At pH 6.8, liposome internalization increased 7 times, with an average intensity of (70.41±4.96). This may be due to the charge reversal effect of DLD in the slightly acidic environment of the tumor. At pH 6.8, the negative charge on the surface of DLD turns into a positive charge, which promotes cellular uptake. The results show that DLD/HYP-Lip is sensitive to pH, specifically increases the uptake of HYP by tumor cells in the tumor microenvironment, and improves the effect of tumor treatment. Figure 2 Cellular uptake (A and B) of DLD/HYP-Lip in CBRH-7919 cells quantitatively measured using FCM. Data are expressed as mean ± SD (n = 3). ** p <0.01.

Figure 2 Cellular uptake (A and B) of DLD/HYP-Lip in CBRH-7919 cells quantitatively measured using FCM. Data are expressed as mean ± SD (n = 3). ** p <0.01.

The cellular uptake pathway was determined, and the results showed that in the presence of the giant pinocytosis inhibitor amiloride (p <0.05), all cellular uptake of DLD/HYP-Lip was significantly reduced, while in the presence of chlorpromazine A similar phenomenon was observed under the circumstances. Clathrin-mediated endocytosis inhibitor. In contrast, the presence of filipin, an inhibitor of caveolin-mediated endocytosis, has no significant effect on the cellular uptake of DLD/HYP-Lip. It is speculated that DLD-Lips will initially enter cells in macropinosomes and clathrin-coated vesicles through macropinocytosis and clathrin-dependent endocytosis, respectively, and further endosome/lysosomes Into the cell. Due to the DLD conjugate containing lysine, DLD-Lips may produce a proton sponge effect similar to polyethyleneimine (PEI), causing endosomes/lysosomes to swell and destroy, thereby making intact liposomes in the cytoplasm. Released. 50,51 It is worth noting that internalization through macropinocytosis is more effective than clathrin-mediated endocytosis, because it is compatible with clathrin-coated nanocarriers in avoiding endosome/lysosome degradation. In contrast, the more porous giant cytoplasmic membrane structure can increase the leakage of inclusion bodies to the cytoplasm. 52,53 The contents of the experiment and the results of "Identification of Cell Uptake Pathways" (Figure S1) are shown in the supporting information.

In order to prove the potential of the prepared mitochondrial targeting liposome DLD/HYP-Lip in tumor treatment, the endosomal escape of the mitochondrial part was studied by confocal fluorescence microscopy. As shown in the supporting information results (Figure S3), the incubation time increases and more particles are released into the cytoplasm (red dots in the merged image), indicating the escape of liposomes from lysosomes. The results show that DLD/HYP-Lip can successfully target mitochondria, and DLD plays a vital role in the targeting process.

Through the MTT test, the cytotoxicity of blank DLD-Lips to CBRH-7919 cells with different lipid concentrations at pH 7.4 and 6.8 was evaluated, and the survival rate of untreated cells at a specific pH was 100%. Figure 3A shows that blank DLD-Lips is non-toxic even at high lipid concentrations under two pH conditions; therefore, DLD-Lips may be a safe drug delivery system. The MTT method is also used to evaluate the cytotoxicity of HYP, SPC/HYP-Lips, and DLD/HYP-Lips to liver cancer CBRH-7919 cells at pH 7.4 and 6.8. At pH 6.8, Figure 3B-D shows that DLD/HYP-Lips is also more cytotoxic to CBRH-7919 cells than HYP and SPC/HYP-Lips. DLD/HYP-Lips promote the death of CBRH-7919 cells. Therefore, these data are consistent with the data from the cell uptake study. In addition, the pH-induced surface charge reversal of liposomes contributes to the internalization of liposomes and the accumulation of anticancer drugs in cells. At the same pH value, the DLD/HYP-Lips IC50 of CBRH-7919 cells is eight times lower than that of HYP. HYP, SPC/HYP-Lips and DLD/HYP-Lips. The IC50 of CBRH-7919 cells were 4.89 μg/mL, 13.82 μg/mL, and 18.08 μg/mL at pH 7.4; HYP, SPC/HYP-Lips and DLD/HYP-Lips IC50 of CBRH-7919 cells were at pH 6.8 They are 4.59 μg/mL, 12.91 μg/mL, 0.26 μg/mL, respectively. Therefore, DLD/HYP-Lips has the greatest inhibitory effect on the growth of CBRH-7919 cells, especially at pH 6.8. This enhanced inhibition can be attributed to higher intracellular uptake that triggers apoptosis by targeting mitochondria. Figure 3 Cytotoxicity of SPC solution (A), HYP solution (B), SPC/HYP-Lip (C) and DLD/HYP-Lip (D) (pH 7.4 and pH 6.8) to CBRH 7919 (mean ± SD, n = 3).

Figure 3 Cytotoxicity of SPC solution (A), HYP solution (B), SPC/HYP-Lip (C) and DLD/HYP-Lips (D) (pH 7.4 and pH 6.8) to CBRH 7919 (mean ± SD, n = 3).

In order to determine whether the enhanced anti-cancer effect of DLD Lips stems from the mitochondrial-mediated apoptosis pathway, mitochondrial targeting was first examined with CLSM and flow cytometry (FCM). Figure 4A shows the CLSM image of DLD/HYP-Lips mitochondrial localization in CBRH-7919 cells. The yellow dots indicate the co-localization of the green fluorescence from Mitotracker Green FM and the red fluorescence from DLD/HYP-Lips. Bright yellow fluorescence indicates that DLD/HYP-Lips selectively accumulates in mitochondria, while SPC/HYP-Lips (no yellow fluorescence) does not. Figure 4 Mitochondrial targeting of liposome formulations. Localization of DLD/HYP-Lips mitochondria in CBRH-7919 cells by confocal laser scanning microscope. CBRH-7919 cells were incubated with SPC/HYP-Lips and DLD/HYP-Lips for 12 hours, and then stained with Mitotracker Green FM. The yellow dots in the merged picture indicate the co-localization of liposomes in the mitochondrial compartment. The scale bar represents 25 μm (A). The cumulative amount of HYP in the mitochondria of CBRH-7919 cells at pH 6.8 measured by flow cytometry (B and C). The activity ratio of caspase 9 (D) and caspase 3 (E). Data are expressed as mean ± SD (n = 3). *p <0.05, **p <0.01.

Figure 4 Mitochondrial targeting of liposome formulations. Localization of DLD/HYP-Lips mitochondria in CBRH-7919 cells by confocal laser scanning microscope. CBRH-7919 cells were incubated with SPC/HYP-Lips and DLD/HYP-Lips for 12 hours, and then stained with Mitotracker Green FM. The yellow dots in the merged picture indicate the co-localization of liposomes in the mitochondrial compartment. The scale bar represents 25 μm (A). The cumulative amount of HYP in the mitochondria of CBRH-7919 cells at pH 6.8 measured by flow cytometry (B and C). The activity ratio of caspase 9 (D) and caspase 3 (E). Data are expressed as mean ± SD (n = 3). *p <0.05, **p <0.01.

Mitochondria are the main place where cells carry out aerobic respiration, and adenosine triphosphate (ATP) is formed through oxidative phosphorylation. 54 Mitochondria are also organelles capable of reproducing in eukaryotic cells. Mitochondria control cell apoptosis, and many lethal signaling pathways are concentrated on mitochondria. 55 The discovery of the mechanism of mitochondria regulating cell apoptosis has made it a broad prospect for anti-tumor targets. 56-58 Studies have shown that the occurrence and development of tumors is apoptosis. Related, mitochondria have a regulatory effect on cell apoptosis. 59,60

Figure 4B and C respectively show the cumulative amount of the drug in the mitochondria of CBRH-7919 cells, expressed by the amount of fluorescence intensity. After the cells were incubated with DLD/HYP-Lips for a period of time, the mitochondrial fluorescence intensity was significantly higher than that in the SPC/HYP-Lips group. The fluorescence intensity is (65.16±5.07), which is about 3.4 times higher than the SPC/HYP-Lips group (18.23±1.59). The above data shows that DLD-Lips can effectively target mitochondria and increase the accumulation of drugs in mitochondria.

The content of HYP in tumor tissue mitochondria was also measured. The results (Figure S2) showed that the content of HYP in tumor tissue mitochondria in the DLD/HYP-Lip administration group was significantly higher than that in the saline group, HYP and SPC/HYP-lip group. The results show that DLD/HYP-Lip can be enriched in the mitochondria of tumor tissues, which enhances the toxicity of DLD/HYP-Lip to tumor tissues. See supporting information for experiment content and results.

The activation of apoptosis-related enzymes is an essential factor in the process of apoptosis. Caspase 9 and caspase 3 are usually used as downstream markers of tumor cell apoptosis. 61-63 Figure 4D and E respectively reflect the caspase 3 activity of caspase 9 and CBRH-7919 cells treated with different HYP preparations. After DLD/HYP-Lips treatment, caspase 9 activity in CBRH-7919 cells was 1.68 times higher than that in the control group, and caspase 3 activity was 1.97 times higher than that in the control group. Compared with other HYP preparations, DLD/HYP-Lips has the most significant effect of inducing caspase 9 and caspase 3 activation. These data indicate that cell death induced by DLD/HYP-Lip is mediated by the mitochondrial-dependent apoptosis pathway.

Early and late apoptotic cells were counted to prove that HYP induces apoptosis of liver cancer CBRH-7919 cells (Figure 5A-E). When using HYP, SPC/HYP-Lip and DLD/HYP-Lip (6.0 mg/mL HYP), the apoptosis induction rates were 24.44%, 10.28% and 41.38%, respectively. In addition, blank DLD-Lip induced apoptosis at a rate of 1.52%, similar to the control group (0.96%). The results show that blank DLD-Lips has no direct effect on cell apoptosis. In addition, DLD concentrates liposomes in cells, exerts cytotoxic effects and promotes cell apoptosis, indicating that cell death induced by DLD/HYP-Lip is mediated by the mitochondrial-dependent apoptosis pathway. Figure 5 Apoptosis of CBRH-7919 cells (AE) treated with culture medium, HYP, SPC/HYP-Lip, DLD/HYP-Lip and DLD-Lip for 12 hours and measured by flow cytometry.

Figure 5 Apoptosis of CBRH-7919 cells (AE) treated with culture medium, HYP, SPC/HYP-Lip, DLD/HYP-Lip and DLD-Lip for 12 hours and measured by flow cytometry.

In order to confirm the feasibility of DLD/HYP-Lips in cancer treatment in vivo, tumor xenograft models were used to evaluate the anti-tumor efficacy of DLD/HYP-Lips in vivo. The anti-tumor effect was reflected by measuring the changes in tumor volume of tumor-bearing nude mice in each test group within 25 days (Figure 6A). The tumors of the nude mice in the saline injection group grew very fast. Both HYP and SPC/HYP-Lip inhibit tumor growth to a certain extent, while DLD/HYP-Lip almost completely inhibit tumor growth, indicating that DLD/HYP-Lip has a significant anti-cancer effect. Figure 6B shows a picture of the tumor on day 25 and the growth curve of the tumor. Figure 6 Anti-tumor efficacy of various HYP preparations in CBRH-7919 tumor-bearing mice. As indicated by the arrow, mice (n = 5) received an injection of HYP (6.0 mg/kg). Changes in tumor growth over time (A); the tumor is photographed at the end of the experiment (B). Separate tumor tissue and calculate tumor growth inhibition (TGI, %) (C); morphological changes of tumor HE stained sections (D); use Ki67 staining to assess tumor cell proliferation in vivo (proliferating cells appear brown (D). Ki67 index calculation It is the ratio of proliferating cells to total cells in each field of view (n = 5) (E). Tumor cell evaluation uses TUNEL assay for apoptosis in vivo (apoptotic cells are shown in brown) (D); percentage of apoptosis index Calculated as the ratio of apoptotic cells to total cells in each field (n = 5) (F). 1. Saline, 2. HYP, 3. SPC/HYP-Lips and 4. DLD/HYP-Lips treatment. The results are expressed Mean ± SD, *p <0.05, **p <0.01. The scale bar represents 250 µm.

Figure 6 Anti-tumor efficacy of various HYP preparations in CBRH-7919 tumor-bearing mice. As indicated by the arrow, mice (n = 5) received an injection of HYP (6.0 mg/kg). Changes in tumor growth over time (A); the tumor is photographed at the end of the experiment (B). Separate tumor tissue and calculate tumor growth inhibition (TGI, %) (C); morphological changes of tumor HE stained sections (D); use Ki67 staining to evaluate tumor cell proliferation in vivo (proliferating cells appear brown (D). Ki67 index calculation It is the ratio of proliferating cells to total cells in each field of view (n = 5) (E). Tumor cells are evaluated for apoptosis in vivo using the TUNEL assay (apoptotic cells are shown in brown) (D); percentage of apoptosis index Calculated as the ratio of apoptotic cells to total cells in each field (n = 5) (F). 1. Saline, 2. HYP, 3. SPC/HYP-Lips and 4. DLD/HYP-Lips treatment. The results are expressed Mean ± SD, *p <0.05, **p <0.01. The scale bar represents 250 µm.

After the experiment, the tumors in each drug group were stripped and weighed, and the tumor inhibition rate was calculated (Figure 6C). The tumor inhibition rates of HYP, SPC/HYP-Lip and DLD/HYP-Lip were 28.78%, 41.71% and 88.79%, respectively. The results show that DLD/HYP-Lip has the best anti-tumor effect.

After the tumor suppression experiment in tumor-bearing nude mice was completed, the tumor tissues were removed for immunohistochemical staining. As shown in Figure 6D, the results of the HE experiment showed that DLD/HYP-Lips caused the largest area of ​​apoptosis in drug-resistant tumor tissues. Ki67 immunohistochemical staining experiments showed that tumor cell proliferation levels in tumor-bearing nude mice injected with DLD/HYP-Lip were significantly reduced (Figure 6E). The TUNEL test showed that tumor-bearing nude mice injected with DLD/HYP-Lip more significantly induced apoptosis of drug-resistant tumor cells (Figure 6F). The reasons for the enhanced efficacy of DLD/HYP-Lip may be as follows: (1) The negatively charged DLD/HYP-Lip in the circulation interacts little with plasma proteins, so it can resist non-specific protein absorption and avoid the rapid elimination of RES. (2) DLD/HYP-Lips with a suitable particle size can induce HYP to accumulate more in tumor tissues (pH 6.8) through the EPR effect, and charge conversion increases the uptake of DLD/HYP-Lips by cells, thereby enhancing the growth of liver cancer cells. Cytotoxicity; (3) The apoptosis-inducing effect of DLD/HYP-Lips can improve the anti-cancer effect.

Healthy tumor-free BALB/c mice are used to evaluate the in vivo toxicity of HYP, SPC/HYP-Lip and DLD/HYP-Lip (6.0 mg/kg HYP). The clinical toxicity behavior and body weight changes of the mice during the administration period were recorded. After the end of the final observation period, the organs were harvested for HE staining experiments. Compared with the control group, there were no obvious animal toxicity-related symptoms such as dehydration, dyskinesia, muscle loss, and anorexia during the administration period. The body weight changes of tumor-bearing nude mice in each group are shown in Figure 7A. The weight of mice in each administration group increased slightly, indicating that there may not be serious systemic toxicity in each administration system. The results of the HE staining experiment showed that the organs of the mice in each administration group did not appear obvious damage (Figure 7B). Figure 7 In vivo toxicity test in normal mice. The body weight curve of mice receiving different treatments (HYP 6.0 mg/kg) (A). The morphological changes of HE staining in different organs of normal mice with different treatments (B). 1. Normal saline, 2. HYP, 3. SPC/HYP-Lip, 4. DLD/HYP-Lip. The scale bar represents 250 µm.

Figure 7 In vivo toxicity test in normal mice. The body weight curve of mice receiving different treatments (HYP 6.0 mg/kg) (A). The morphological changes of HE staining in different organs of normal mice with different treatments (B). 1. Normal saline, 2. HYP, 3. SPC/HYP-Lip, 4. DLD/HYP-Lip. The scale bar represents 250 µm.

In this study, a dual-function tumor-targeted liposome (DLD-HYP-Lips) was developed, in which HYP is delivered to the mitochondria of tumor cells to enhance anti-cancer activity. Unlike most therapeutic liposomes that function at the cellular level, our DLD-Lips loaded with HYP target mitochondria at the subcellular level to achieve more effective treatment. The results indicate that DLD-Lips can be directed to mitochondria by modification with the pH-responsive phospholipid DSPE-Lys-DMA. Compared with free HYP and traditional liposomes loaded with HYP, DLD-Lips loaded with HYP increased the cytotoxicity and in vivo anti-tumor effects of liver cancer CBRH-7919 cells. This is due to increased cell uptake and mitochondrial-mediated apoptosis, which significantly reduces the mitochondrial membrane potential, activates caspase-9/3 and induces apoptosis. Therefore, DLD-Lip is a promising anti-cancer drug delivery system that can be used for cancer treatment in vitro and in vivo.

This research was funded by the National Natural Science Foundation of China (81703944), the Natural Science Foundation of Heilongjiang Province (YQ2019H031), the Heilongjiang Provincial Research Startup Fund (2020), and the Heilongjiang Provincial Excellent Innovative Talents Project of the University of Traditional Chinese Medicine (2018).

The authors report no conflicts of interest in this work.

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