Journal of Nanomedicine & Biotherapeutic Discovery
Open Access

Inhibition of Hepatocellular Carcinoma Cell Proliferation and Metastasis by Levorotatory Polylactic Acid Electrostatically Spun Nanofibers Loaded with Ibuprofen and Acid-Sensitive Adriamycin Hydrochloride Mesoporous Silica

Yanxu Li¹, Weiben Ji¹, Zhuoheng Liu², Lai Chang⁵, Huiyuan Hu², Kunlin Wu¹, Ke Wang², Chaoying Wang¹, Quan Zhang³, Jiefeng Gao⁴, Tao Wang^3* and Wei Wu^{1 *}Correspondence: Tao Wang, Department of Veterinary Medicine, Yangzhou University, Yangzhou, China, Email:

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Introduction

Primary Hepatocellular Carcinoma (HCC) has become the fifth most common cancer in the world and its incidence is increasing, accounting for 90% of primary liver tumors in China [1,2]. As the early onset of HCC is insidious, most patients should not be aware of it, which leads to the fact that most patients are in the progression stage upon detection and the available treatments are very limited [3,4]. Treatment for HCC patients usually depends on the severity of the disease and currently liver transplantation, surgical resection and localized radiofrequency ablation are the main treatments, but only 30% of the patients are eligible for treatment and the medium and long-term survival rates of postoperative patients are very low [5,6]. However, only 30% of patients are eligible for treatment and the medium and long-term survival rates of postoperative patients are still low [7,8]. Patients taking palliative hepatic resection in the middle and late stages have a high postoperative recurrence rate, metastasis occurs earlier and the postoperative 5- year survival rate is lower; at the same time, due to its postoperative chemotherapeutic side effects and toxicity, it leads to poor patient compliance and the therapeutic efficacy does not meet expectation. In the process of tumor proliferation, infiltration and metastasis, the inflammatory factors IL-1β, IL-6 and other regulatory factors such as TNF-α, MMP and Reactive Oxygen Species (ROS) can change the tumor microenvironment, degrade the basement membrane and promote tumor angiogenesis as well as residual tumor invasion. And in tumors, PCNA, Bcl2/Bax ratio, CXCR4/CXCL12 are highly correlated with tumor invasion and metastatic ability. Therefore, improving the tumor microenvironment, especially targeting some inflammatory factors, may be efficacious in inhibiting tumor progression, postoperative recurrence and curing certain tumors.

Adriamycin hydrochloride (DOX) is an anthracycline cytotoxic drug that has been shown to have in vitro cytotoxic effects on primary cultures of a wide range of identified tumors [9]. The antiproliferative and cytotoxic effects of DOX derive from the following [10]. DOX binds to DNA to form a complex, which in turn inhibits the synthesis of DNA, RNA and proteins. The embedding process also triggers topoisomerase II cleavage of DNA, producing a cytocidal effect. DOX also inhibits DNA helicase activity, preventing DNA double-strand unraveling and interfering with replication and transcription. DOX can participate in redox reactions by generating cytotoxic free radicals. Despite the fact that DOX is the cornerstone of HCC chemotherapy, DOX remains ineffective in monotherapy or in combination with other chemotherapeutic agents [11,12]. Therefore, the mechanism of DOX resistance must be investigated to provide a basis for further clarification of therapeutic strategies for HCC [13].

Functional blood vessels within the tumor are often unable to meet the demands of the expanding tumor cells within the tumor, resulting in a lack of oxygen and other nutrients. Subsequent production of lactic acid in the absence of oxygen and hydrolysis of ATP in the absence of energy leads to the formation of an acidic environment around the tumor, which is directly neutralized by Sodium Bicarbonate (SB). Tumors are known as "hard-to-heal wounds" and the different types of inflammatory cells surrounding them can secrete a variety of immunomodulatory factors that promote tumor development [14]. Ibuprofen (IBU) inhibits Cyclooxygenase (COX) activity in vivo, which in turn reduces Prostaglandin (PG) biosynthesis in local tissues and also inhibits the expression of certain cellular mucosal molecules, thus suppressing inflammation in various ways [15]. Narona M, et al. [16], found that oleanolic-acid-oxime conjugated ibuprofen and ketoprofen compounds have significant effects on the signaling pathways associated with HCC, which can be used as potential targets for new drug design and help to reduce the invasiveness and chemoresistance of hepatocellular carcinoma cells.

Nanoparticles with diameters around 100 nm can mimic natural structures and biological functions and MSNs are widely used as drug carriers because of their huge specific surface area and pore volume, uniform and adjustable size distribution of pore sizes, easy chemical modification of the inner and outer surfaces and excellent biocompatibility. Polylactic Acid (PLA) can be absorbed in vivo and has a wide range of applications in biomedicine. Its stereoisomeric form, Levulinic Polylactic Acid (PLLA), is a synthetic material based on α-hydroxyl groups that is biocompatible and does not cause an immune response in the body. PLLA has a wide range of applications in biomedicine and is by far the most promising biodegradable synthetic material [17-19].

Therefore, in order to treat residual tumors after HCC surgery and prevent postoperative recurrence in HCC patients, we designed a drug carrier system that can rapidly release antiinflammatory drugs to control the spread of inflammatory factors in the early stage after hepatectomy, continuously release chemotherapeutic drugs in the late stage to prevent tumor cells from residual to HCC recurrence after surgery and at the same time, can be used as a scaffold for tissue growth and repair. This drug carrier system utilizes mesoporous silica (MSN) loaded with adriamycin hydrochloride (DOX) and Sodium Bicarbonate (SB) and the drug-carrying MSN, Ibuprofen (IBU) and levorotatory Polylactic Acid (PLLA) were spun into nanofibrous membranes by electrostatic spinning. It was aimed at local treatment after HCC surgery and the prevention of residual tumor recurrence.

Materials and Methods

Materials

Mesoporous silica (MSN) was purchased from Nanjing Kike Nano Bio Co., Ltd, China (Item No. JK-04-010-100), adriamycin hydrochloride (DOX), Ibuprofen (IBU), levorotatory Polylactic Acid (PLLA) was purchased from Aladdin Biochemistry and Technology Co., Ltd, Shanghai, China (CAS:25316-40-9, CAS: 15687-27-1, CAS :33135-50-1), Sodium Bicarbonate (SB) was purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd, China (CAS:144-55-8), Annexin VFITC/ PI apoptosis detection kit was purchased from Nanjing Novozymes (Item no.: A211-01), liver cancer cells HepG2 and Huh7 were obtained from the Chinese Academy of Sciences (Shanghai, China) cell bank. dMEM medium and fetal bovine serum were purchased from GIBCO (Item no.: 11960044, 12483020).

Drug-loaded and acid-sensitized processes in mesoporous silica

15 mg of DOX was added to a methanol-water solution with a volume ratio of 3:5, mixed well and then 200 mg of MSN was added and stirred at 37°C for 24 h. After centrifugation at 12,000 rpm for 15 min, the supernatant was retained. The supernatant was washed twice with the above ratio of methanol to water solution, each time centrifuged at 12000 rpm for 5 min and the supernatant was retained. MSN@DOX was added to 2 ml of a saturated aqueous solution of Sodium Bicarbonate (SB) at 20°C and stirred for 24 h. The supernatant was retained by centrifugation at 12000 rpm for 15 min. The precipitate obtained above was lyophilized.

Drug loading rate

The DOX supernatant was mixed thoroughly, the absorbance at 234 nm was measured by a UV spectrophotometer and the drug loading capacity was calculated according to the formula Absorbance A=0.0063 × Concentration C (ug/ml)-0.0006. The supernatant of sodium bicarbonate was added with acetone according to acetone:supernatant=5:2 (V:V). SB was precipitated and dried by centrifugation and weighed.

Electrostatic spinning

MSN@DOX and MSN@DOX-SB containing 200 mg of MSN were ultrasonically dispersed into a mixture of dichloromethane, hexafluoroisopropanol and ethanol in a mass ratio of 5:2:2 and PLLA, DOX and IBU were stirred until dissolved. The fibrous membranes were spun under electrospinning conditions at a voltage of 20 kV, a flow rate of 0.04 ml/min and a receiver distance of 15 cm and the resulting fibrous membranes were labeled as PLLA-DOX, PLLA-MSN@DOX, PLLA-MSN@DOXSB, PLLA-MSN@DOX-IBU and PLLA-MSN@DOX-SB-IBU respectively.

Transmission electron microscopy (HRTEM)

MSN@DOX, MSN@DOX-SB were characterized by Tecani G2 F30 field emission transmission electron microscope (FETEM), USA.

Scanning Electron Microscopy (SEM)

The resulting fiber membranes were characterized using a Japanese S-4800 II field emission scanning electron microscope. The fiber membrane was placed in liquid nitrogen for 3 minutes and the fiber cross section was photographed after extraction.

Compositional analysis and particle size analysis

HRTEM was used for compositional analysis. Ten MSNs and nanofibers were randomly selected from the above characterization photos and their diameters were measured using ImageJ and analyzed by Graphpad Pism 9.5.

In vitro drug release experiments

In a 10 cm cell culture dish with 7 ml of sterile PBS (pH=7.0 and pH=5.0), 1 mg of MSN@DOX, 1 mg of MSN@DOX-SB, as well as 1 × 1 cm of PLLA-DOX, PLLA-MSN@DOX, PLLAMSN@ DOX-SB, PLLA-MSN@DOX-IBU and PLLAMSN@ DOX-SB-IBU fiber membranes were placed in the above two different pH PBS solutions and their UV absorbance at 224 nm and 234 nm were measured at fixed time points 2 h, 4 h, 6 h, 12 h, 24 h and the following 40 days after the residual placement and their concentrations were calculated and release curves were plotted.

Cell lines

Human hepatocellular carcinoma cell lines HepG2, Huh7 and myoblast C2C12 cell lines were obtained from the Chinese Academy of Sciences (Shanghai, China) cell bank. All cell lines were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin/penicillin (Yeasen, Shanghai, China). The constant temperature cell culture incubator was set at 37 °C and the CO₂ concentration was 5% for culture.

Cytotoxicity assay

Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation and cytotoxicity. C2C12, HepG2 and Huh7 cells were trypsin digested and counted and the cell suspension was inoculated into 96-well plates; each well contained about 1000 cells and 100 ul of culture medium was added to the culture for 24 hours. Add 90 ul of medium containing DOX, MSN@DOX, MSN@DOX-SB, MSN@DOX+IBU and MSN@DOX-SB+IBU and after soaking the fiber membrane for 72 hours, incubate for 12 hours. Replace 90 ul of new medium after 48 and 72 hours and add 10 ul of CKC-8 solution to each well and incubate in the incubator at 37°C for 4 hours. The absorbance at 450 nm was determined by enzyme markers.

Apoptosis detection

Cells were inoculated in 6-well plates, and after the number of cells per well reached 2×105, the medium containing DOX, MSN@DOX, MSN@DOX-SB, MSN@DOX+IBU and MSN@DOX-SB+IBU was added to the wells. After soaking the fiber membranes for 72 hours, the medium was incubated for 12 hours, 48 hours and 72 hours. Cells were digested with EDTA-free trypsin and centrifuged at 1,800 rpm (300 × g) at 4°C for 5 min and the supernatant was discarded. Cells were washed twice with pre-cooled PBS and centrifuged each time at 1,800 rpm (300 × g) at 4°C for 5 min. 100 μl of 1 × Binding Buffer from the Annexin V-FITC/PI apoptosis detection kit was added and gently blown to a single-cell suspension. Add 5 μl Annexin V-FITC and 5 μl PI staining solution, gently blow well; incubate for 10 min at room temperature (20 ~ 25°C), protected from light; add 400 μl 1 × binding buffer, gently mix well. The stained samples were detected by flow cytometry within 1 hour. The Annexin V-FITC/PI apoptosis detection kit was purchased from Vazyme (A211-01).

Cell migration assay

C2C12, HepG2 and Huh7 cells were trypsin-digested into single-cell suspensions in a 37°C, 5% CO₂ incubator for 24 hours. After the cells completely covered the 6-well plate, a 200- ul sterile lance tip was used to scratch perpendicular to the 6- well plate and the drawn straight line. After the scratches were centered and perpendicular as observed by the microscope, the cells were washed with sterile PBS three times and serum-free DMEM medium immersed in fibrous membrane for 72 hours was added and samples were taken and photographed at the time points of 0, 12 and 24 hours, respectively. Six horizontal lines were randomly scratched by the Image J software and the cells were then incubated in a 5% CO2 incubator at 37°C for 24 hours. The mean cell spacing and cell mobility were calculated. Cell migration rate=(initial mean cell-to-cell distance-mean cellto- cell distance at time t)/initial mean cell-to-cell distance.

Determination of Reactive Oxygen Species (ROS)

Cells were inoculated in a 6-well plate and after the cells lay flat on the bottom, the medium was replaced with medium impregnated with fiber membrane for 12, 24 and 48 hours, then DCFH-DA was diluted with serum-free culture solution according to 1:1000, so that the final concentration was 10 μmol/L. The cells were incubated for 20 minutes at 37 °C in a cell culture incubator. Remove the cell culture medium and add 1 ml of diluted DCFH-DA. Incubate for 20 min at 37°C in a cell culture incubator. The cells were washed three times with serumfree cell culture medium and the fluorescence intensity was measured by enzyme markers and observed by fluorescence microscopy.

Western blot experiment

Cells were lysed using RIPA lysate containing protease inhibitors and lysed proteins were added to SDS loading buffer at 100°C, boiled, denatured, separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Merck, Germany). Membranes were closed in 5% skimmed milk and incubated with a specific primary antibody overnight at 4°C, followed by incubation with a secondary antibody for 2 h at room temperature. Proteins on the membrane were visualized using electrochemiluminescence (Thermo Fisher Scientific, USA) and signals were detected using a ChemiDocTM MP imaging system (BioRad Laboratories, California, USA). Antibodies used in Western blotting: Anti-Bax antibody, anti- Bcl2 antibody, anti-CXCR4 antibody, anti-Caspase3 antibody, anti-PCNA antibody, anti-COX2 antibody and anti-bate actin antibody were purchased from Abcam.

qPCR experiment

Total RNA was extracted using Trizol (Thermo Fisher Scientific, USA) and the isolated total RNA was quantified and transcribed into cDNA using a reverse transcription kit (TOLOBIO, Shanghai, China). 2-ΔΔCq method was used to calculate the quantification using GAPH as a control. RT-PCR was used to detect the mRNA expression level of the test genes. The primers were designed and synthesized by bioindustry according to NCBI gene information and the specific primer sequences are shown in Table 1.

Table 1: Primer information for RT-Qpcr.

ELISA experiment

Cells were gently washed with cold PBS, trypsin digested, centrifuged at 1000 × g for 5 min and collected. The collected cells were washed with cold PBS three times, resuspended by adding 150 μL-200 μL of PBS to every 106 cells and the cells were broken by sonication. Centrifugation was performed at 1500 × g for 10 min at 2°C-8°C and the supernatant was retained. Add 100 ul of standard working solution or sample to the well plate, incubate at 37°C for 90 min, discard the liquid and then add 100 ul of biotinylated antibody working solution, incubate at 37°C for 60 min, discard the liquid and wash with PBS for 3 times. Add 100 ul of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 min, discard the liquid and wash with PBS for 5 times. Add 90 ul of substrate solution to each well. The wells were incubated at 37℃ for 15 minutes, 50 ul of termination solution was added and 450 nm was detected.

Statistical analysis

Each experiment was repeated independently at least three times. Continuous variables were expressed as the mean and Standard Deviation (SD). Continuous variables were compared using an unpaired two-tailed Student's t-test or one-way ANOVA. All analyses were completed using GraphPad Prism 9.5 software (GraphPad Software, USA). p-values<0.05 were considered statistically significant. Asterisks "^*", "^**", "^***" and "^****" represent P<0.05, respectively, P<0.01, P<0.001 and P<0.0001, respectively.

Results

Transmission electron microscopy and scanning electron microscopy pictures

In MSN@DOX and MSN@DOX-SB transmission electron micrographs, the nanoparticles are hollow mesopores with uniform diameters and the red parts are visible to be decomposed after the electric field emission for the SB. Scanning electron microscope photographs of nanofiber membranes, the fibers are visible to be raised for the nanoparticles inside. Scanning electron microscopy of the cross section of the nanofiber membrane after liquid nitrogen extraction shows that internal nanoparticles are visible inside PLLA-MSN@DOX. The above results indicate that the nanoparticles are successfully dispersed into PLLA and that the nanofibers contain nanoparticles inside.

Compositional analysis, loading rate and particle size analysis

Transmission Electron Microscopy (TEM) compositional analysis revealed that the main component of MSN@DOX was silica and Na+ was visible in MSN@DOX-SB. The OD values were measured three times and the drug loading mass of DOX was calculated from the absorbance equation to be about 8.9904 ± 0.08919 mg, with a drug loading rate of about 59.936%. Acetone: supernatant=5:2 (V:V) The precipitate was dried and weighed at 35 mg after the addition of acetone; the solubility of SB at 30°C was 9.6 mg/ml; and the calculated drug loading mass of SB was 35 mg. The diameters of the nanoparticles and nanofibers were measured at 10 randomly taken photographs of different characterizations; the diameter of the nanoparticles was 97.094 ± 5.354 nm using Inage J and the diameter of the nanofibers was 1.903 ± 0.139 um.

In vitro release experiments

Based on their release curves, it can be concluded that there was no significant difference in the release rates of MSN@DOX and MSN@DOX-SB at pH=7, while the release rate of DOX from MSN@DOX-SB increased at pH=5. The release rate of DOX from the PLLA-DOX nanofiber membranes was faster at pH=5.0, which shows that the acidic environment was favorable for dispersion. The release rate of DOX in PLLA-MSN@DOX and PLLA-MSN@DOX-SB was faster in the acidic environment compared to that in PLLA-MSN@DOX-SB. The release rate of IBU and DOX in PLLA-MSN@DOX-IBU and PLLAMSN@ DOX-SB-IBU nanofibrous membranes was faster in an acidic environment than in a neutral environment. Release rates in an acidic environment were faster than those in a neutral environment. In conclusion, it can be seen that the acidic environment is favorable for drug release from nanofibers.

Nanofiber membrane has lower biotoxicity

DOX can lead to oxidative stress and the death of cardiomyocytes and it has a certain degree of myocardial toxicity [20]. According to the results of CCK8 experiments, it is known that DOX alone produces greater damage to the cells, with higher cytotoxicity. The use of DOX-carrying nanocarrier systems can significantly reduce the toxicity of DOX on C2C12 cells and the toxicity of DOX is further reduced by further wrapping the nanoparticles through electrostatic spinning. DOX has a certain degree of tumor inhibitory effect and is clinically applied to a wide range of tumors, but due to the obvious side effects, the patient's compliance is poor. Nanoparticles and nanofibrous membranes can release DOX slowly, which reduces the side effects of the drug and at the same time, plays a tumor inhibitory role. The release rate of DOX in MSN is faster compared with that of the nanofibrous membrane and the tumor inhibitory effect is stronger in the short term. However, the culture medium after soaking the fiber membrane for 15 days also showed more obvious tumor inhibition, while SB did not show obvious cytotoxicity, although it could regulate the pH of the medium. IBU has a certain tumor inhibitory effect, which was confirmed in prostate cancer, but the addition of IBU to MSN did not show any enhancement of the tumor inhibitory effect, so no obvious cytotoxicity was seen for IBU.

Nanofiber membrane promotes apoptosis of tumor cells and has no obvious effect on normal cells

According to the CCK8 results, it is known that DOX has a certain toxic effect on C2C12 cells and hepatocellular carcinoma cells Huh-7 and Hep3B cells. For this reason, we verified the effects of different factors on the apoptosis of three kinds of cells. Compared with DOX alone, the apoptosis ratio of cells after encapsulation by nanoparticle loading and PLLA did not differ from that of the control group (p<0.05), suggesting that the toxicity of DOX on myofibroblasts was reduced after encapsulation. In the inhibitory effect on tumor cells, we found that there was a tumor inhibitory effect in the medium after 15 days of immersion in nanoparticles or fibrous membranes and the apoptosis rate increased significantly after 72 hours compared with the control group, which further indicated that nanoparticles and nanofibrous membranes were able to promote apoptosis of tumor cells and had a certain tumorsuppressing effect, but no significant pro-apoptotic effect was seen by the addition of SB and IBU to the MSN.

In summary, it can be seen that DOX has greater toxicity to both normal myofibroblasts and hepatocellular carcinoma cells (Huh-7 and Hep3B). DOX has significantly reduced toxicity after being loaded into nanoparticles of MSN and further encapsulated by PLLA, but it still has a tumor-inhibitory effect. SB and IBU do not have any significant effect on normal cells and do not show any significant inhibitory effect on tumor cells, so we can rule out the effect of SB and IBU on the tumor cells. Although the toxicity of MSN@DOX and MSN@DOX-SB was weakened, they still had some cytotoxicity due to the fast initial release rate, so we chose nanofibrous membranes for the subsequent verification of tumor inhibitory effects.

Nanofiber membrane can inhibit tumor cell invasion

The cell scratch assay was mainly applied to the effects of drugs on cell migration, repair and interactions. DOX is cytotoxic and usually inhibits cell migration. DOX is more toxic to tumor cells and has a low rate of cell migration (P<0.0001). When it was loaded into MSN and further wrapped by PLLA, cell migration and repair increased, but the rate of migration and repair was still low compared with that of the negative control group (P<0.0001), which further indicated that DOX still had an antitumor effect although it was loaded into MSN and further wrapped by PLLA.

Nanofibrous membranes can promote elevated ROS levels in tumor cells

An important factor in tumorigenesis and response to anticancer therapies is the regulation of oxidative stress. There may be direct or indirect regulation of ROS levels in signaling pathways associated with tumorigenesis. High levels of ROS are damaging to normal cells, but in tumor cells, ROS levels are usually elevated, mainly as a result of metabolic and signaling aberrations that lead to alterations in the redox state within tumor cells 10. In our study, we found that nanoparticles and nanofibrous membranes elevated reactive oxygen species levels in Hep3B cells compared to controls, while some studies have found that excessively high ROS levels have an inhibitory effect on tumor cells 12 and with Huh-7 cells we got the same conclusion.

Nanofibrous membranes can attenuate the expression of inflammatory factors, improve the tumor microenvironment, promote HCC cell apoptosis and inhibit the proliferation and metastasis of HCC cells

PLLA-MSN@DOX-SB-IBU reduces COX-2 expression and promotes HCC cell apoptosis: COX-2 expression in HCC is associated with decreased overall survival and disease-free survival and therefore a poorer prognosis. Direct neutralization of lactic acid produced by tumor cells in a hypoxic environment, improvement of the acidic environment and inhibition of the secretion of immunomodulatory factors by inflammatory cells around the tumor and thus inhibition of COX-2 activity in vivo, can improve the prognosis of HCC patients. It has been shown that SB and IBU have significant effects on HCC-related signaling pathways 16. In Hep3B cells, it was verified by WB and qPCR experiments that the DOX group could reduce COX-2 levels better than the other groups, which might be related to the greater toxicity of DOX, leading to massive cell death. PLLAMSN@ DOX-SB-IBU in each nanofibrous membrane could better reduce the COX-2 level, mainly due to the fact that the drug was still continuously released over time and the tumor inhibition effect was gradually enhanced, so this was also the reason for the inhibition of COX-2 expression. The results of the WB and qPCR experiments in Huh-7 cells were the same as those of the experiments in Hep3B cells.

PLLA-MSN@DOX-SB-IBU reduces TNF-α expression and inhibits metastasis of HCC cells: Hepatocyte Growth Factor (HGF) responds to macrophage secretion of TNF-α, which plays an important role in malignant metastasis. Both TNF-α and HGF overexpression in the microenvironment are closely associated with poor HCC differentiation, suggesting that inflammation in the hepatic microenvironment may promote microvascular invasion in HCC 24. DOX, on the other hand, is the cornerstone of HCC chemotherapy, with its antiproliferative and cytotoxic effects. Studies have shown that DOX has in vitro cytotoxic effects on primary cultures of a wide range of identified tumors and can lead to a significant reduction in the level of TNF-α in HCC 25. According to the ELISA results of Hep3B cells, compared with the negative control group and DOX group, each nanofiber membrane had a TNF-α lowering effect (p<0.0001). With the continuous release of the drug in the fiber membrane, the expression of TNF-α was gradually reduced in each fiber membrane and the most significant reduction in TNF-α expression was observed in the PLLAMSN@ DOX-SB-IBU group (p<0.0001) and the TNF-α expression was reduced in the 72-month period (p<0.0001). (0.0001) and the decrease of TNF-α expression at 72 h was not different from that of the DOX group. The results of the ELISA experiments on Huh-7 cells were the same as those on Hep3B cells.

PLLA-MSN@DOX-SB-IBU reduces IL-1β and IL-6 expression and inhibits HCC cell proliferation and metastasis: IL-1β and IL-6 are key cytokines driving the development of chronic liver diseases and they play an important role in the development of HCC due to their direct effect on host inflammation. It has been shown that IL-6 and IL-1β can upregulate the expression of proliferation-related proteins in HCC cells through multiple signaling pathways, which in turn promote the infiltration and metastasis of HCC 26. IL-1β and IL-6-related signaling pathways show a positive correlation with HCC cell viability, proliferation, invasion, migration and drug resistance, so inhibition of the expression of IL-1β and IL-6 can increase apoptosis of HCC cells. The results of ELISA analysis of Hep3B cells showed that each nanofiber membrane had the effect of decreasing L-6 and IL-1β compared with the negative control group and the DOX group (p<0.0001), among which PLLAMSN@ DOX-SB-IBU had the most significant decrease in the expression level of both (p<0.01). The results of the Hep3B cell experiment were the same.

PLLA-MSN@DOX-SB-IBU promotes apoptosis and inhibits cell proliferation more than other nanofiber membranes

PLLA-MSN@DOX-SB-IBU elevates Bax/Bcl2 and promotes apoptosis in HCC cells: The Bax gene is the most important apoptosis gene in the human body and belongs to the Bcl-2 gene family and the encoded Bax protein can form a heterodimer with Bcl-2 to inhibit Bcl-2. The overexpression of Bax/Bcl-2 is closely related to the pathological grade and survival of HCC. The expression of Bcl-2 in tumor cells inhibits the adhesion, spreading and movement of cells through the enhancement of actin polymerization. Studies have confirmed the finding that high Bax/Bcl-2 expression is a potent predictor of HCC prognosis. According to the experimental results, the elevated level of Bax expression and the reduced level of Bcl2 expression were most significant in the DOX group compared to the control group (p<0.0001). The release of DOX from each nanofiber membrane increased with time, the expression of Bax increased and the expression of Bcl2 decreased, and the increase of Bax (p<0.05) and the decrease of Bcl2 were more obvious (p<0.01) in the PLLA-MSN@DOX-SB-IBU group compared with the other nanofiber membrane groups in the Hep3B cells. At 72 h, the expression of Bax and Bcl2 was increased in the PLLAMSN@ DOX-SB-IBU group compared with the control group (p<0.0001). The level of Bcl2 reduction in the PLLAMSN@ DOX-SB-IBU group was not significantly different from that in the DOX group. Therefore, PLLA-MSN@DOX-SB-IBU could promote the elevation of Bax expression and the reduction of Bcl2 expression, thus elevating the Bax/Bcl2 ratio, which further indicated that PLLA-MSN@DOX-SB-IBU could better promote apoptosis of HCC cells. The results of WB and qPCR experiments in Huh-7 cells were the same as those in Hep3B cells.

PLLA-MSN@DOX-SB-IBU elevates Caspase3 expression and promotes apoptosis in HCC cells: Apoptosis is a recognized mechanism of cell death in cancer therapy. It has been shown that tumor cells use the apoptotic process to generate effective growth-stimulating signals to stimulate the regeneration of tumors undergoing radiotherapy. And Caspase3 is a key executor of apoptosis 32, as it is partially or wholly involved in proteolytic cleavage of many key proteins, such as the nuclease Poly (Adp) Ribose (PARP) Polymerase, which further initiates the cascade mechanism of caspases that in turn leads to apoptotic cell death. Therefore, higher amounts of activated Caspase3 in tumor tissues were associated with significantly increased recurrence and mortality rates. The expression level of Caspase3 in Hep3B cells at 12 h was not significantly different from the negative control, differed significantly from that of the DOX group (p<0.0001) and was further increased with the increased release of DOX at 72 h. At 72 h, the expression of Caspase3 in the PLLA-MSN@DOX-SB-IBU group was differentially reduced from that of the DOX group and the expression of Caspase3 in the PLLA-MSN@DOX-SB-IBU group was the highest among the four types of nanofiber membranes, which could significantly elevate the expression of Caspase-3 and thus play a role in promoting the programmed apoptosis of cells. Huh-7 cells. The results of WB and qPCR experiments were the same as those of Hep3B cells.

PLLA-MSN@DOX-SB-IBU inhibits the CXCL12-CXCR4 axis and inhibits HCC progression: CXCR4 and its ligands play critical roles in disease and cancer. CXCL12 is dependent on intra- and extracellular conditions and precisely regulates signaling through the activation of CXCR4, which in turn is involved in disease progression. Chemokines and their receptor CXCL12-CXCR4 axis are thought to be important factors in the regulation of angiogenesis, which is essential for the growth and development of hepatocellular carcinoma. Tumor-derived DNA can be recognized by immune cells, thus inducing an autoimmune response, but when tumor-derived DNA passes through the CXCL12-CXCR4 axis, it reduces apoptosis, resulting in immune escape. Jeng et al. demonstrated that through the CXCL12-CXCR4 signaling pathway, which further suggests that the CXCL12-CXCR4 axis can stimulate cancer cell migration. Based on the results of qPCR experiments, it was known that both DOX and nanofiber membrane groups could reduce the expression of CXCL12 (p<0.05) and at 72 h in each nanofiber membrane group, PLLA-MSN@DOX-SB-IUB reduced the expression of CXCL12 more significantly in Huh-7 cells (p<0.001). In Hep3B cells, PLLA-MSN@DOX-SB-IUB reduced the expression of CXCL12 more significantly and the difference with the DOX group was smaller (p<0.05). According to the analysis of the results of WB experiments, it can be seen that the expression level of CXCR4 in the PLLA-MSN@DOX-SB-IUB group was reduced more significantly in Hep3B cells compared with other nanofiber membrane groups and there was no significant difference with the DOX group at 72 h. The results of WB and qPCR experiments in Huh-7 cells were the same as those in Hep3B cells. Therefore, PLLA-MSN@DOX-SB-IUB can better reduce the expression of CXCL12 and CXCR4, promote tumor cell apoptosis and inhibit tumor cell migration compared with other nanofiber membranes.

PLLA-MSN@DOX-SB-IBU can reduce PCNA expression and HCC cell proliferation: Hepatocyte proliferation disruption is an important pathophysiologic process in hepatocarcinogenesis. The proliferation of cancer cells, including HCC cells, has been shown to be closely related to Proliferating Cell Nuclear Antigen (PCNA). Studies have shown that PCNA is highly expressed in hepatocellular carcinoma tissues, promotes carcinogenesis and progression and has been recognized as an important prognostic biomarker for determining the survival of HCC patients. Evidence suggests that PCNA regulates proliferation by acting as a scaffold to recruit proteins involved in DNA replication, DNA repair, cell cycle control and survival and chromatin assembly. qPCR and WB experiments showed that the level of PCNA reduction by each nanofiber membrane group was not as pronounced as that of the DOX group, but the PLLAMSN@ DOX-SB-IUB group reduced PCNA expression levels more significantly than the other nanofiber membrane groups and the PLLA-MSN@DOX-SB-IUB group reduced PCNA expression levels in Hep3B cells at 72 h. The results of WB and qPCR experiments on Huh-7 cells were the same as those on Hep3B cells. The Hep3B cell experimental results were the same. Therefore, PLLA-MSN@DOX-SB-IUB can effectively inhibit PCNA trimerization and further limit the proliferation and tumor growth of HCC cells.

Discussion

Due to the high rate of HCC postoperative recurrence and the absence of an effective means of prevention, we successfully prepared PLLA nanofibrous membranes by using the MSNloaded chemotherapeutic drug DOX and by using SB acid sensitization of MSN@DOX and further dispersing MSN and IBU into PLLA for electrostatic spinning in anticipation of their role in inhibiting tumor progression and postoperative recurrence. We used CCK8 experiments and apoptosis experiments to verify that DOX cytotoxicity was reduced after being loaded by MSN and encapsulated by PLLA. There was no obvious toxic effect on myofibroblast C2C12, but there was still a certain pro-apoptotic effect on tumor cells. After adding SB and IBU to MSN, CCK8 experiments showed no obvious effect on normal cells and no obvious inhibitory effect on tumor cells, so we can exclude the effect of SB and IBU on tumor cells. In the comparison of each nanofibrous membrane with a negative control and DOX as a positive control, we found that PLLAMSN@ DOX-SB-IBU could better reduce the secretion of inflammatory factors in tumor cells, inhibit the proliferation of tumor cells and promote apoptosis. This nanofibrous membrane loaded with SB can neutralize the acidic microenvironment formed by tumor cells due to high oxygen consumption and in addition, IBU can assist DOX in reducing the expression of inflammatory factors, thus enhancing the tumor inhibitory effect of DOX. Since PLLA, MSN@DOX-SB and IBU were electrostatically spun into nanofibrous membranes, they can be implanted into the tumor resection site after surgery, thus further inhibiting its possible tumor cells, thus achieving the effect of preventing tumor recurrence by continuously releasing drugs to inhibit the possible residual tumor cells after surgery, which brings hope to the patients applying chemotherapeutic drugs against tumor recurrence after surgery.

Conclusion

In addition, the buried diaphragm can be performed immediately after surgery, which is easy to operate and brings convenience to clinical treatment. Although we have completed the preparation of the nanofiber membrane and conducted a controlled study on its cytotoxicity to verify the feasibility of this material as a drug carrier system, its toxicity study on the important organs of the body as well as experimental animals still needs to be improved. In follow-up, we will establish an in situ transplantation tumor palliative hepatectomy model in nude mice with highly metastatic human hepatocellular carcinoma to evaluate the tumor-suppressive effect and safety of the dual drug-carrying composites in vivo.

Acknowledgments

This article is supported by the National Natural Science Foundation of China (Project number, 82001964) and Yangzhou University Medical Innovation and transformation Special fund (Project number, AHYZUCXTD202104).

Disclosure

The author(s) report no conflicts of interest in this work.

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