Drug-loaded MITO@HA nanodrugs for evaluating the efficacy of targeted therapy for pancreatic cancer

Drug-loaded MITO@HA nanodrugs for evaluating the efficacy of targeted therapy for pancreatic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Drug-loaded MITO@HA nanodrugs for evaluating the efficacy of targeted therapy for pancreatic cancer Fengjun Liu, Zhiyang Xu, Xiaochao Jia, Yidan Tang, Mingsheng Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3972887/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The purpose of this study was to evaluate the efficiency of mitoxantrone hydrochloride (MITO@HA) as a targeted pancreatic cancer therapy. MITO@HA binds to sodium hyaluronate, which is highly expressed in pancreatic cancers. This study seeks to evaluate MITO@HA treatment efficacy, clarify its inhibitory effect on pancreatic cancer, and provide an experimental basis for the use of organic polymer nanoparticles loaded with antitumor drugs to treat pancreatic cancer. This treatment strategy was developed for pancreatic cancer based on the hydrophobic behavior of the nanopharmaceutical MITO@HA. The average particle size of MITO@HA was 51.4 ± 2.3 nm, and the particles had a spherical structure. CCK-8 assays revealed that both MITO and MITO@HA inhibited the proliferation of pancreatic cancer cells. that the most suitable experimental conditions were determined to be exposing pancreatic cancer cells to 0.5 uM/L MITO@HA for 2 days. PANC-1 pancreatic cancer cells and pancreatic cancer tissues were found to express high levels of CD44. In in vitro experiments, MITO@HA inhibited G0/G1 phase arrest, increased apoptosis, and decreased cell replication, cell migration and invasion in the pancreatic cancer cell cycle compared to MITO alone. Therefore, we believe that MITO@HA has a good tumor cell inhibitory effect. Furthermore, in vivo experiments revealed that the tumor volume in nude mice in the MITO@HA group decreased (P < 0.05), and both MITO and MITO@HA treatment decreased the tumor growth curves, with MITO@HA decreasing them more than MITO alone. Compared with those in the control group and the MITO group, the HE staining of tumors in the MITO@HA group showed massive liquefaction necrosis of the tumor tissues. Safety evaluation of the nude mice in the MITO@HA group revealed that the mice had a normal blood profile, normal liver and kidney function, and normal myocardial enzymes. The above results indicate that MITO@HA can effectively accumulate in pancreatic cancer tumor tissue through the EPR effect and CD44 receptor targeting, leading to liquefaction and necrosis of tumor tissue, thereby effectively reducing tumor growth. The above results showed that MITO@HA is highly safe and can enhance the antitumor effect on pancreatic cancer, providing an experimental basis for clinical application. MITO@HA CD44 Nanomedicine carriers Pancreatic cancer Antitumor effect High security Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Pancreatic cancer is one of the most lethal digestive system malignancies and is both highly aggressive and metastatic, accounting for more than 200,000 deaths per year 1–3 . Due to the lack of typical early-stage clinical manifestations, diagnosis is difficult, and 80% of patients are diagnosed at an advanced stage, limiting the efficacy of surgery 4 . Therefore, chemotherapy plays a very important role in treating pancreatic cancer. Gemcitabine, cisplatin, and paclitaxel are the major current therapeutic options for pancreatic cancer, but their use is limited by strong toxicity and low therapeutic efficacy. 5–7 In addition, a number of potent small molecules targeting PDK are important milestones in the history of cancer research, including for example, dichloroacetic acid (DCA) and its derivatives, dihydroxyphenylpyrazole M77976, hordenine, quercetin, and the sulfone AZD7545 (which showed potent antiproliferative activity in different cancer cell lines and mouse models). Unfortunately, because of their low potency or poor selectivity, none of these small molecules have succeeded in passing clinical trials 8–10 . Clinical trial data are still lacking, and additional studies are urgently needed to verify the relevant effects and to develop more personalized treatment approaches. To improve the current situation, researchers have developed a variety of polymer nanoparticles as drug carriers, generally approximately 10 ~ 200 nm in diameter, which use their various advantages, including targeting and stimulating response release, to effectively improve the therapeutic efficacy against tumors 11–14 . Improving the survival rate of pancreatic cancer patients has always been a difficult problem for domestic and foreign scholars. Due to the poor drainage of lymph fluid in pancreatic cancer patients, which results in high permeability and long retention and targeting effects, nanodrugs of suitable size can enter tumors through relatively small blood vessels with relatively tight intercellular spaces, stay in tumors for several days, both passively and actively accumulate at relatively high concentrations 15–17 . The study of polymer nanoparticles for the treatment of pancreatic cancer is currently uncommon and is still in the exploratory research stage. Hyaluronic acid (HA) is an acidic mucopolysaccharide that is the main component of the extracellular matrix, and its structure is a repeating unit composed of D-glucuronic acid and N-acetylglucosamine, which are water soluble, biocompatible, and biodegradable 18–24 . More importantly, hyaluronic acid also targets the CD44 receptor 25 , which can accumulate and aggregate in a variety of malignant tumor tissues overexpressing the CD44 receptor, increasing the concentration of local drugs; therefore, it has been widely studied and used in the field of nanodrug delivery. Mitoxantrone hydrochloride (MITO), which binds to DNA molecules, inhibits nucleic acid synthesis, and causes cell death, is a nonspecific drug used for the cell cycle 26–27 . Some studies have shown that mitoxantrone has serious side effects, such as myelosuppression and cardiotoxicity, which seriously impede the use of its conventional formulations in the clinic 28–30 . In recent years, to enhance MITO’s antitumor effects and reduce its toxic side effects, MITO research has mainly focused on targeted drug delivery systems. Based on previous research 31 , this paper constructed a MITO@HA nanodrug synthesized with sodium hyaluronate as a targeted drug carrier and wrapped it with mitoxantrone hydrochloride to evaluate the antitumor effect on pancreatic cancer (Scheme 1 ) 2. Materials and Methods 2.1. Materials Cell culture reagents: fetal bovine serum (HyClone, Cat.No.SH30087.01), RPMI-1640 medium (HyClone, Cat.No.SH30809.18), DMEM-high sugar medium (HyClone, cat.No.SH30019.01), penicillin (HyClone, Cat.No. SH30010), PBS potassium phosphate buffer (HyClone, Cat. No. SH30256.01B), an Annexin V-FITC apoptosis assay kit (Keygen, No. KGA106), a cell cycle assay kit (Keygen, No. KGA511), a Transwell cell culture plate (BD, No. REF353097), a Matrigel gel (BD, No. 356234), and mitoxantrone hydrochloride (MCE, No. HY-13502A) were used. A microplate reader (Thermo Fisher Scientific No. Multiscan MK3) and an ELISPOT (AID iSpot system) were used. A Suzhou Antai Clean Workbench (SW-CJ-IFD), a low-speed centrifuge (Zhongjia, SC3614), an inverted light microscope (OLYMPUS CKX41, U-CTR30-2), a cell thermostatic incubator (Thermo Scientific, HERACELL150i), an inverted fluorescence microscope (Leica, No. DMI6000B), flow cytometry (BD Calibur), a camera system (Guangzhou Mingmei Technology Co., Ltd., No. MC30), and software analysis (Java, No. ImageJ 1.44) were used. SPF-grade BALB/C-NU nude mice were purchased from Guangdong Pharma Biotechnology Co., Ltd. 2.2. Expression of CD44 in pancreatic cancer cells First, 0.5 ml of 0.25% pancreatic enzyme without EDTA was added to PANC-1 pancreatic cancer cells and incubated until cells were observed under a microscope to begin to detach from the culture plate wall. The cells were gently resuspended in medium from step 1 or prechilled with 1×binding buffer to a density of approximately 1×10 6 cells/mL. Then, 0.5 mL of the cell suspension from the cell culture plate (5×10 5 cells) was transferred into a clean centrifuge tube. After adding FITC mice transferrin to the anti-human CD44 antibody and incubating it in the dark for 30 min, the cells were centrifuged with PBS 3 times, immediately analyzed via flow cytometry. The sterilized 20 mm coverslips were placed in a 90 mm dish, the cells were seeded in the dish at a density of 2×10 4 /ml PANC-1 pancreatic cancer cells for cell crawling, and immune cell and histochemical staining was performed 5 days later. The cells were washed with PBS three times for 2 min each And then fixed in 4% paraformaldehyde for 15 min (cold methanol:acetone 1:1) and air-dried for 5 min. The cells were then washed three times with PBS for 2 min each. The cells were incubated with 0.5% Triton X-100 (with DPBS) once for 20 minutes. The cells were washed with PBS three times for 2 min each. The mixture was incubated with 3% H2O2 for 15 min. The cells were washed with PBS three times for 2 min each. Samples were then incubated with blocking serum (5% normal secondary antibody serum DPBS solution) for 20 min, followed by primary antibody incubation (PBS with titers 1:200, wet box) at 40°C overnight or at 37V for 60 min. The negative control was cleaned with primary antibody-derived serum and PBS three times for 5 min each. The secondary antibody working solution was incubated with PV6001 (wet box) at 37°C for 30 min. The specimens were washed 5 times with PBS for 2 min each. DAB was used for color development (protected from light, observed to brown under the microscope) for approximately 10 ~ 15 minutes. Finally, the samples were washed once in distilled water for 5 min, counterstained with hematoxylin for 10 min, and then washed once again in distilled water for 5 min. Afterwards, the samples were sealed and all photos collected. 2.3. Preparation of the nanoprobe of MITO@HA 300 mg of sodium hyaluronate was weighed with a balance, dissolved in 50 ml of deionized water, and stirred for 10 min to dissolve it thoroughly. Then, 30 mg of mitoxantrone hydrochloride was weighed, dissolved in 5 ml of deionized water until fully dissolved, added dropwise to sodium hyaluronate solution (3–4 drops/min), and stirred overnight. The extra free drug was removed by dialysis (MWCO 3500 Da) for 24 hours, after which the solution was lyophilized to obtain the MITO@HA powder. All the procedures were performed in the dark. The particle size, drug load (DLC), and drug carrying rate (DLE) of MITO@HA were determined via ultraviolet spectroscopy and inductively coupled plasma atomic emission spectrometry. DLC and DLE are calculated using the following formulas: DLC(wt.%)= (mass of nanoparticle-loaded drug) / (total amount of drug - mass of nanoparticles)×100% DLE(wt.%)= (mass of nanoparticle-loaded drug) / (mass of total drug input) ×100% 2.4. Cell culture and grouping PANC-1 pancreatic cancer cell lines were cultured in 1640 culture medium supplemented with 10% fetal bovine serum and 1% biclonal antibody and subcultured in an incubator with 5% CO2 saturated humidity and 37°C. The cells were subpassaged, cultured in 12-well plates and divided into control groups, MITO groups, and MITO@HA groups (6 complex wells per group). 2.5. Cytotoxicity assay The CCK-8 method was used to incubate pancreatic cancer cells for 1 d in combination with MITO@HA (0 µg/ml, 1 µg/ml, 2.5 µg/mL, 5 µg/mL, 20 µg/mL, 50 µg/mL). Additionally, seven different concentrations of 0 µmol/L, 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L, 1 µmol/L, 2 µmol/L and 4 µmol/L were added to the control group, MITO group and MITO@HA groups, respectively. After digesting each group of cells, the cell count was adjusted to 1×10 5 cells/well, divided into 96-well plates, and 100 µL wells, that is, 1×10 4 cells/well. Adherent cells were allowed to adhere, after which the cells were collected at each time point for detection. Cells (0 h, 24 h, 48 h, 72 h) were collected at various time points and added to CCK-8 solution (Keygen Bio, Cat.No. KGA317), the scale is 1/10. Briefly, 100 µl of culture solution was added to 10 µl of detection solution. After 4 h of incubation, the microplate reader was used to read the plate, and the OD450 data were obtained via the CCK-8 assay. 2.6. Cell cycle The cells in the control group, MITO group, and MITO@HA group were incubated with 0.5 µmol/L PBS, MITO, or MITO@HA for a total of 2 days and incubated with 0.25% pancreatic enzyme without EDTA until microscopic observation that the cells had begun to detach from the culture plate wall. The cells were collected by centrifugation, the supernatant was discarded, the cells were washed twice with precooled PBS, precooled 70% ethanol was added, and the cells were fixed overnight at 4 ℃ or fixed for a long time at -20 ℃. The cells were centrifuged and collected, washed with 1 mL of PBS once, 500 µl of PBS containing 50 µg/mL propidium bromide (PI), 100 µg/mL LRNaseA, and 0.2% Triton X-100 was added, and the mixture was incubated in the dark at 4°C for 30 minutes. Using standard procedures, flow cytometry was used to detect and count 20000 to 30000 cells. The results were analyzed using the cell cycle fitting software ModFit. 2.7. Cell apoptosis The cells in the Control, MITO, and MITO@HA groups were incubated for 2 days in MITO@HA supplemented with 0.5 ml of 0.25% pancreatic enzyme without EDTA until the cells were observed to detach from the plate wall via microscopy. The cells were gently resuspended in medium from step 1 or prechilled with 1× binding buffer to a density of approximately 1×10 6 cells/mL. 0.5 mL of the cell suspension was then transferred from the cell culture plate (5×10 5 cells) into a clean centrifuge tube. Then, 1.25 µL of Annexin V-FITC was added. The mixture was protected from light for 15 min at room temperature (18–24°C). The mixture was centrifuged at room temperature (1,000×g) for 5 min to remove the supernatant. The cells were gently resuspended in 0.5 ml of prechilled 1×binding buffer. 10 µL of propidium iodide was added to the sample solution. The samples were stored on ice protected from light. The cells were then detected via flow cytometry for analysis. 2.8. Cell cloning The cells in the control group, MITO group, and MITO@HA group were incubated with 0.5 µmol/L PBS, MITO, and MITO@HA for a total of 2 days, cells from the different groups were collected via trypsinization, and the cells were counted in 1 ml of medium. Pipette into a single-cell suspension, count the cells, dilute the cells into 1×10 4 cells/ml, seed in a 6-well plate, and pipette 300 µl seeded to 300 cells/well. After inoculation, each well was supplemented with medium to 2000 µl/well. The plate was shaken back and forth horizontally to ensure that the cells were evenly distributed in the wells. Every day, when a single cell grew into a clump-like clone, generally after 7 days of culture, each well of medium was removed, the cells were rinsed with PBS or normal saline 2 times, and each well was supplemented with 200 µl of crystal violet staining solution to fully cover the bottom of the well. After 20 min, the 96-well plate was rinsed with tap water, the cells were allowed to proliferate, and the flow of water was decreased. After rinsing, the number of colonies was calculated. Analyses, scans and photographs were taken using an enzyme-linked spot image automatic analyzer. 2.9. Cell migration and invasion The cell culture plate Control group, MITO group, and MITO@HA group were each incubated for 2 days with 0.5 µmol/L PBS, MITO, MITO@HA, respectively, cells from the treatment group were collected, 1×10 5 cells were counted, resuspended with 100 µl serum-free medium, added to the upper chamber of the Transwell cell culture plate, and 600 µl complete medium was added to the lower chamber. The cell culture plates were CO 2 incubated at 37°C, 5% CO 2 for 12–48 h, taken out the chamber, and the cells in the upper chamber were wiped off with a cotton swab. Cells were then fixed in 4% paraformaldehyde for 20 min, wash with PBS once, were crystal violet stained for 10 min, and then washed with PBS again. Afterwards, the cells were observed under the microscope to determine whether the cells passed through the small wells. If the cells passed through other experimental groups, images were taken for statistical analysis. Upper ventricular lower surface cells stained with crystal violet were eluted with 33% acetic acid, after which the OD570 was determined via a microplate reader. The Matrigel was dissolved overnight at 4°C, diluted with prechilled serum-free medium at a volume ratio of 1:3, 40 µl was added to the prechilled Transwell chamber, and the mixture was incubated at 37°C for 2 h to solidify the Matrigel. The excess liquid in the chamber was aspirated, 100 µl and 600 µl of serum-free medium were added to the upper chamber and lower chamber, respectively, and the cells were equilibrated at 37°C overnight. One day after the drugs were added, 1×105 cells were counted, the cells were resuspended in 100 µl of serum-free medium, the Transwell chamber was added to the upper chamber, and 600 µl of complete medium was added to the lower chamber. After incubation at 37°C and 5% CO 2 for 24 and 48 hours, the cells were taken out the chamber, the cells in the upper chamber were wiped off with a cotton swab, and then fixed 4% paraformaldehyde for 15 min. Afterwards, they were washed once with PBS, stained with crystal violet for 10 min, washed again with PBS. If the cells pass through the small wells and through other experimental groups, pictures for statistics were. Upper ventricular lower surface cells stained with crystal violet were eluted with 33% acetic acid, after which the OD570 was determined via a microplate reader. 2.10. Tumor Inhibition Studies Five-week-old male nude mice received 0.1 ml (containing 5×107 PANC-1 cell suspension in nude mice for subcutaneous tumorization). The tumor-bearing nude mice were randomly divided into 3 groups: the control group; the MITO group, and the MITO@HA group (n = 3 per group). The tumors of tumor-bearing nude mice were allowed to grow to 3-4mm, and the mice all received intravenous injection of PBS, MITO, or MITO@HA at a 10 mg/kg equivalent dose, which amounted to an injection dose of 100 µl. The mice were injected every three days for a total of 3 times. Mouse tumor volume was measured every 3 days. On Day 26 after tumoring, the mice were sacrificed by inhalation of carbon dioxide, after which the tumors were harvested and weighed. 2.11. Toxicity assessment Pancreatic cancer tumor-bearing nude mice were divided into three groups: control group, MITO group and MITO@HA group (n = 3 per group). Tumor-bearing nude mice were intravenously injected with PBS, MITO, or MITO@HA at a 10 mg/kg equivalent dose, and 100 µl was injected once every three days for a total of 3 injections. On the 26th day after tumor seeding, the mice were sacrificed by inhalation of carbon dioxide, and eye blood was collected to detect blood parameters, liver and kidney function, and cardiac enzymes for safety assessment. 2.12. Statistical analysis The statistical results of the measurement data are expressed as the mean ± standard deviation (mean ± SD), and the statistical analysis and graphing software used SPSS and GraphPad Prism. After satisfying the normality distribution and homogeneity of variance tests, the two groups of data were compared via an independent t test, and the comparisons between multiple groups of data were performed via one-way ANOVA. P < 0.05 indicates that the difference is statistically significant; * indicates P < 0.05, ** indicates P < 0.01, **** indicates P < 0.001, and **** indicates P < 0.0001. 3. Results and Discussion 3.1. Expression of CD44 in pancreatic cancer cells In recent years, nanotechnology has become one of the most important technologies at present, playing an important role in the rapid development of several research fields, especially in the field of precision cancer treatment. HA has good biocompatibility and CD44 receptor targeting 32-33 , which can accumulate and aggregate in PANC-1 pancreatic cancer tumor tissues overexpressed by CD44 receptor, target the release of chemotherapy drugs, and improve anti-tumor efficiency. Flow cytometry to determine CD44 expression levels in PANC-1 pancreatic cancer cells. The results (Figure 1A) showed that 99.9% of pancreatic cancer cells significantly overexpressed CD44.Pancreatic cancer cells (Figure 1B) and tissue immunohistochemistry(Figure 1C) showed high expression of CD44. 3.2. Constructing MITO@HA 3.2.1 Characterization of MITO@HA In this study, we successfully created an MITO@HA-targeted nanomedicine through the hydrophilic interaction of HA with MITO, and we evaluated its therapeutic efficacy on pancreatic cancer cells in vitro and on pancreatic cancer animals in vivo via tail vein injection in nude mice to investigate its targeted inhibitory effect on pancreatic cancer. Compared with traditional chemical drugs, polymer nanoparticle-loaded antitumor drugs offer advantages: (1) targeted therapy 34 and (2) enhanced tumor accumulation 35 . MITO@HA nanodrugs can enhance the antitumor efficacy and reduce toxic side effects of these drugs by actively targeting CD44Rs and enhancing the permeability and retention (EPR) effect on the surface of pancreatic cancer tumors. First, solutions at different concentrations (10 μg/ml, 15 μg/ml, 25 μg/ml, 35 μg/ml, 50 μg/ml, 75 μg/ml) were prepared with a mitoxantrone hydrochloride standard aqueous solution, and the ultraviolet absorption spectrum was determined via an ultraviolet spectrophotometer, with 610 nm as the maximum absorption peak. The linear relationship between the concentration of the drug and the absorbance was determined as a standard curve (Figure 2A and Figure 2B). By measuring the absorbance of MITO@HA at 610 nm, the encapsulation rate and drug loading were calculated to be 45.82±1.72% and 4.15±0.82%, respectively (Figure 2C). The authors demonstrated the successful self-assembly of nanoparticles between hyaluronic acid and mitoxantrone through electrostatic interactions. This study provides a basis for subsequent active targeted therapy for pancreatic cancer. Next, the morphological characteristics and particle size distributions of the nanoions were analyzed. MITO@HA The average hydrated particle size measured by a dynamic light scattering nanolaser was 51.4 ± 2.3 nm, and the particle size in the TEM image was consistent with the particle size measured by particle size analysis. Moreover, the surface of MITO@HA had high electrostatic stability (-26.1 ± 3.2 mV) (Figure 2D). 3.2.2 Screening of the MITO@HA concentration After treating pancreatic cancer cells with 1 μg/ml, 2.5 μg/mL, 5 μg/mL, 20 μg/mL, and 50 μg/mL MITO, as determined by the CCK-8 method, it was found that the concentration of MITO@HA significantly inhibited the proliferation of cells in the MITO group (Figure 2E). When the concentration was increased to μmol/L, the efficacy and toxicity of seven different concentrations of MITO@HA were evaluated in pancreatic cancer cells after 0, 1, 2 and 3 days. MITO@HA was incubated with pancreatic cancer cell precursors at a concentration of 0.5 μmol/L for 2 days at the most appropriate concentration and time (Figure 2F). 3.3 In vitro study In the present study, inhibition of pancreatic cancer cells by MITO@HA for 2 days was most suitable for treating pancreatic cancer cells at a concentration of 0.5 μmol/L. Compared with those in the MITO group, the cells in the MITO@HA group exhibited cell cycle arrest at the G0/G1 phase, increased apoptosis, decreased cell replication, cell migration and invasion inhibition. 3.3.1 Cell cycle The cell cycle results (Figure 3A) showed that the average percentages for each group were 24.91±1.42%、39.37±1.27%和42.67±0.944%, respectively, in the Control group, MITO and MITO@HA groups, indicating that nanoparticles affected the cycling activity of PANC-1 pancreatic cancer cells, resulting in most cells in G0/G1 phase blockade (P<0.0001). 3.3.2 Cell apoptosis The apoptosis assay results revealed that (Figure 3B) the average percentages of the sum of the total apoptosis rates (early apoptosis rate Q4 and late apoptosis rate Q2) in the control, MITO, and MITO@HA groups were 3.01±0.12%, 5.30±0.49% and 7.28±0.59%, respectively (P<0.0001). MITO@HA had the greatest impact on the apoptosis of PANC-1 pancreatic cancer cells. 3.3.3 Cell migration and invasion The results of cell migration and invasion are shown in Figure 3C. The average migration and invasion rate of PANC-1 cells in the control group was 280.75±18.51%, which is consistent with the rapid metastasis of pancreatic cancer. The mobility rates of the patients in the MITO@HA and MITO groups were significantly lower than those in the control group (P<0.0001), with 154.50±9.02% and 145.63±7.15%, respectively. Moreover, there was no statistically significant difference between the MITO group and the MITO@HA group (P=0.3539). Similarly, cell invasion was significantly lower in the MITO@HA and MTO groups than in the control group (P<0.0001). The above results showed that MTO has a significant inhibitory effect on tumor growth. In addition, hyaluronic acid (HA) targets the CD44 receptor in pancreatic cancer, and HA-coated MTO can also tightly adhere to the CD44 receptor and target it to inhibit the migration and invasion of tumor cells. 3.3.4 Cell cloning The cell cloning results showed that (Figure 3 D) for the control, MITO, and MITO@HA groups, the average percentages of cell clones in each group were 100.00±3.56%, 47.02±9.53%, and 45.02±7.38%, respectively. The number of clones of PANC-1 pancreatic cancer cells in the MITO@HA group was the smallest and was significantly lower than that in the control and MITO groups. Therefore, we believe that hyaluronic acid (HA) targets the CD44 receptor in pancreatic cancer and plays the most important role in killing pancreatic cancer cells. In addition, the constructed MITO@HA nanodrugs target and concentrate in the plasma of pancreatic cancer cells and release MITO, resulting in significant blockade of the G0/G1 cell cycle in pancreatic cancer, promotion of tumor cell apoptosis, significant reduction of cell cloning, and inhibition of cell migration and invasion. 3.4. In vivo study The results of animal experiments showed that MITO@HA nanodrugs had a significant inhibitory effect on pancreatic tumors. A tumor growth curve was generated for the nude mice with pancreatic cancer (Figure 4A). The growth of the tumors in the control group was the fastest, and the growth of the tumors in the MITO@HA group was slower than that in the control group, indicating that the tumor inhibition effect of the former was significant. The average tumor mass of the nude mouse tumors in each group (Figure 4B) was 0.846±0.016 g in the control group and 0.790±0.0346 g in the MITO@HA group, and the average tumor mass in the MITO@HA group was 0.455±0.529 g (P<0.0001). To further validate the antitumor efficacy of the targeting liposomes, tumor tissue was fixed, sectioned, and HE stained (Figure 4C). Compared with those in the control and MITO groups, the tumor cells in the MITO@HA group exhibited obvious large areas of liquefied necrosis, whereas the control group exhibited no obvious necrosis. The above results showed that MITO@HA had an obvious inhibitory effect on tumors and had good therapeutic effects on tumors. The reason for this difference is that MITO@HA can allow more drugs to reach tumor tissue through EPR effects, and HA can fuse with cell membranes to swallow lipids into cells, increasing tumor cell uptake. In vivo antitumor experiments further proved that surface-modified HA can further promote the accumulation and uptake of MITO@HA at the tumor site through passive and active targeting to better suppress tumor growth. 3.5. In vivo toxicity assessment The biochemical indices of the nude mice in each preparation group are shown in Figure 5. Important parameters such as white blood cells, red blood cells, PLT, ALT, AST, TBIL, ALB, CK, CK-MB, LDH, LDB1, URES, CREA, and UA in the MITO@HA group were normal, indicating that the MITO@HA treatment had good safety. In this study, the biosafety of MITO@HA nanoparticles was further confirmed by blood biochemical indices and enhanced antitumor effects compared to those of the MTIO group. In summary, MITO@HA nanoparticles targeting the CD44 receptor can effectively enhance the therapeutic efficacy against pancreatic cancer tumors, and the safety of these nanoparticles is reliable, suggesting the possibility of clinically targeted treatment for pancreatic cancer. 4. Discussion In recent years, nanotechnology has become one of the most important technologies, playing a crucial role in the rapid development of various research fields, especially in the field of precision cancer treatment. Hyaluronic Acid (HA) exhibits good biocompatibility and CD44 receptor targeting specificity 11–13 .Accumulating in pancreatic cancer tissues with overexpressed CD44 receptors, targeted release of chemotherapy drugs can enhance the anti-tumor efficiency. In this study, hyaluronic acid (HA) was chosen as the raw material, and through hydrophilic and hydrophobic interactions, MITO was loaded to synthesize hyaluronic acid MITO@HA nano-drugs. The study aimed to verify and explore its targeted inhibitory effect on pancreatic cancer by intravenous injection of MITO@HA in nude mice with pancreatic cancer.。Compared to traditional chemotherapy drugs, anti-tumor drugs loaded onto polymer nanoparticles have advantages: ①Targeted therapy 14 ,②Enhanced tumor accumulation 15 .The MITO@HA nano-drug can enhance its anti-tumor efficacy through active targeting to the surface CD44 receptors of pancreatic cancer tumors and the enhanced permeability and retention (EPR) effect, thereby reducing toxic side effects. In this study, pancreatic cancer cell experiments showed that the toxicity and safety of MITO@HA were most suitable when treating pancreatic cancer cells for 2 days at a concentration of 0.5 µmol/L. Compared to the MITO group, MITO@HA exhibited G0/G1 phase arrest in the cell cycle, increased apoptosis, reduced cell cloning, and inhibited cell migration and invasion.The results of animal experiments show that the MITO@HA nano-drug has a significant inhibitory effect on pancreatic tumors. This study further confirmed the biocompatibility of MITO@HA nanoparticles through the detection of blood biochemical indicators, demonstrating its ability to effectively reduce damage to animals caused by chemotherapy drugs. Additionally, MITO@HA enhances the anti-tumor effect of chemotherapy drugs.In summary, MITO@HA nanoparticles, with CD44 receptor targeting specificity, can effectively reduce systemic drug toxicity while enhancing the therapeutic effect on pancreatic cancer tumors. This offers a potential avenue for clinical targeted treatment of pancreatic cancer. 5. Conclusions Based on the characteristics of CD44 receptors, which are highly expressed in pancreatic cancer tumor cells, MITO@HA, which can target the delivery of antitumor drugs, was prepared by using HA to specifically bind to CD44 receptors. MITO@HA was characterized, and the results showed that MITO@HA had a uniform particle size distribution. The results of cell-based experiments showed that MITO@HA-free MITO could significantly increase uptake in pancreatic cancer cells; inhibit tumor cell proliferation, apoptosis, migration, and invasion; and cause cell cycle arrest at the G0/G1 phase. The results of in vivo antitumor experiments proved that MITO@HA can effectively inhibit the growth of tumors and have good antitumor effects, and the animal biochemical indices indicated that they were normal. In summary, HA can target and modify nanodrugs, thereby increasing the targeting efficiency of nanodrugs and improving the antitumor efficiency and safety of MITO@HA. Declarations Authors’ Contributions: Fengjun Liu and Yu Xinshi conceived and designed the study. Tianyou Chen wrote the manuscript. Zhiyang Xu and rewriting the manuscript. Shi Xiudong, Zhiyang Xu, Jia Xiaochao, Yidan Tang, Mingsheng Chen, Chuan Chen, and Fang Fang and interpreted the data. All authors read and approved the final manuscript. Funding: The research was supported by the National Natural Science Foundation of China (Grant 82172029, 82302265), the Shanghai Municipal Health Commission (Grant 202140084), Shanghai Pujiang Program (Grant 21PJD062), the Shanghai Municipal Health Commission, Key discipline construction project of Shanghai Three-year Action Plan for Public Health System Construction (Grant GWV-10.1-XK09)，the National Natural Science Foundation of China (Grant 82302265) , and Shanghai Sailing Program (Grant 21YF1436600). The funder had no role in study design, data collection, analysis, the decision to publish, or the preparation of the manuscript. Conflicts of Interest The authors declared no conflicts of interest in this work. Data availability The dataset presented in the study is available on request from the First author (Fengjun Liu) during submission or after publication. The data are not publicly available due to privacy. Ethics statement Shanghai Public Health Clinical Center Laboratory Animal Welfare & Ethics committee is the animal ethics committee that reviewed our study. The relevant work of the Shanghai Public Health Ethics Committee follows the "Regulations on the Management of Experimental Animals" issued and implemented by the National Science and Technology Commission, and complies with relevant Chinese laws and regulations References Vincent A, Herman J, Schulick R, Hruban R H, Goggins M,et al.Pancreatic cancer［J］.Lancet. 2011,378(9791):607-620. Wael R,Abd-Elgaliel,Ching-Hsuan Tung,et al.Selective detection of Cathepsin E proteolytic activity[J]. Biochim Biophys Acta. 2010,1800(9):1002-1008. Moffat GT, Epstein AS, OReilly EM, et al. Pancreatic cancer-A disease in need: optimizing and integrating supportive care[J]. Cancer,2019, 125 (22):3927-3935． Hue JJ, Sugumar K, Markt SC, et al. Facility volume-survival relationship in patients with early-stage pancreatic adenocarcinoma treated with neoadjuvant chemotherapy followed by pancreatoduodenectomy[J]. Surgery, 2021, 6060 (22):30838-30841. Tao S, Zhao X, Zhang X, Guan X, Wei J, Yuan B, He S, Zhao D, Zhang J, Liu Q, Ding Y. The role of macrophages during breast cancer development and response to chemotherapy[J]. Clin Transl Oncol. 2022, 22: 1938-1951. Shao FY, Wu YF, Tain ZY, Liu SQ. Biomimetic nanoreactor for targeted cancer starvationtherapy and cascade amplificated chemotherapy[J]. Biomaterials. 2021,274:120869. Ju CY, Wen YJ, Zhang LP, Wang QQ, Xue LG, Shen J, Zhang C. Neoadjuvant Chemotherapy Based on Abraxane/Human Neutrophils Cytopharmaceuticals with Radiotherapy for Gastric Cancer[J]. Small. 2019,15(5):1804191. Stacpoole, P.W. Therapeutic Targeting of the Pyruvate Dehydrogenase Complex/Pyruvate Dehydrogenase Kinase (PDC/PDK) Axis in Cancer. J. Natl. Cancer Inst. 2017, 109, 1–14. Wang, X.; Shen, X.; Yan, Y.; Li, H. Pyruvate dehydrogenase kinases (PDKs): An overview toward clinical applications. Biosci. Rep. 2021, 41, BSR20204402. Groaz, E.; De Jonghe, S. Overview of Biologically Active Nucleoside Phosphonates. Front. Chem. 2021, 8, 616863. Peer D, Karp JM, Hong S, FaroKhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy[J]. Nat Nanotechnol. 2007,2(12):751-60. SchroederA,Heller DA,Winslow MM,etal.Treating metastatic cancer with nanotechnology[J]. Nat Rev Cancer, 2011, 12(1): 39-50. Jeevanandam J, Barhoum A, Chan YS, et al.Review on nanoparticles and nanostructured materials: history, sources, toxicity, and regulations[J].Beilstein J Nanotechnol, 2018, 9:1050-1074. Jaidev LR, Chede LS, Kandikattu HK, et al.Theranostic nanoparticles for pancreatic cancer treatment[J]. Endocr Metab Immune Disord Drug Targets, 2021,21(2): 203-214. Zi YX, Yang KY, He JH, Wu ZM, Liu JP, Zhang WL. Strategies to enhance drug delivery to solid tumors by harnessing the EPR effects and alternative targeting mechanisms[J]. Adv Drug Deliver Rev. 2022,114449. He JY, Li CC, Ding L, Huang YN, Yin XL, Zhang JF, Zhang J, Yao CJ, Liang MM, Pirraco RP, Chen J, Lu Q, Baldridge R, Zhang Y, Wu MH, Reis RL, Wang YL. Tumor Targeting Strategies of Smart Fluorescent Nanoparticles and Their Applications in Cancer Diagnosis and Treatment[J]. Adv Mater. 2019,31(40):1902409. Chen WH, Sun Z, Lu LH. Targeted Engineering of Medicinal Chemistry for Cancer Therapy: Recent Advances and Perspectives[J]. Angew Chem int Edit. 2021,60(11):5626-5643. Vasvani S, Kulkarni P, Rawtani D. Hyaluronic acid: A review on its biology, aspects of drug delivery, route of administrations and a special emphasis on its approved marketed products and recent clinical studies[J]. Int J Biol Macromol. 2020,151:1021-1029. Kobayashi T, Chanmee T, Itano N. Hyaluronan: metabolism and function[J]. Biomolecules, 2020, 10(11): 1525. Passi A, Vigetti D. Hyaluronan as tunable drug delivery system[J]. Adv Drug Deliver Rev. 2019, 146: 83-96. Wang R, Huang XB, Zoetebier B, Dijkstra PJ, Karperien M. Enzymatic co-crosslinking of star-shaped poly (ethylene glycol) tyramine and hyaluronic acid tyramine conjugates provides elastic biocompatible and biodegradable hydrogels[J]. Bam. 2023, 20: 53-63. Yan K, Feng YC, Gao K, Shi XJ, Zhao XB. Fabrication of hyaluronic acid-based micelles with glutathione-responsiveness for targeted anticancer drug delivery[J]. J Colloid Interf Sci. 2022, 606: 1586-1596. Liu ZX, Lin WJ, Liu Y. Macrocyclic Supramolecular Assemblies Based on Hyaluronic Acid and Their Biological Applications[J]. Accounts Chem Res. 2022. 55(23): 3417-3429. Camacho KM, Kumar S, Menegatti S, Vogus DR, Anselmo AC, Mitragotri S. Synergistic antitumor activity of camptothecin-doxorubicin combinations and their conjugates with hyaluronic acid[J]. J Control Release. 2015,210:198-207. Garvalho BG, Vit FF, Garvalho HF, Han SW, Delatorre LG. Recent advances in co-delivery nanosystems for synergistic action in cancer treatment[J]. J Mater Chem B. 2021. 9(5): 1208- 1237. Zhang Q, Radvak P, Lee J, Xu Y, Dao VC, Xu M, Zheng W, Chen CZ, Xie H, Ye YH. Mitoxantrone modulates a heparan sulfate-spike complex to inhibit SARS-CoV-2 infection[J]. Sci Rep. 2022. 12(1): 6294. Yan Q , Wang L , Song F , et al. Effects of QDs@Gd 3+ -NGR on targeted fluorescence-magnetic resonance imaging and inhibition of pancreatic cancer cells[J]. Journal of Materials Research, 2020, 35(6):1-9. Heidari Majd M, Asgari D, Barar J, et al. Specific targeting of cancer cells by multifunctional mitoxantrone-conjugated magnetic nanoparticles [J]. Journal of Drug Targeting, 2013, 21(4):328-340. Sargazi A, Shiri F, Keikha S, et al. Hyaluronan magnetic nanoparticle for mitoxantrone delivery toward CD44-positive cancer cells [J]. Colloids and Surfaces B: Biointerfaces, 2018, 171:150-158 Rossato LG, Costa VM, de Pinho PG, et al. The metabolic profile of mitoxantrone and its relation with mitoxantrone-induced cardiotoxicity [J]. Archives of Toxicology, 87(10):1809-1820. Bano F, Banerji S, Howarth M, Jackson DG, Richter RP. A single molecule assay to probe monovalent and multivalent bonds between hyaluronan and its key leukocyte receptor CD44 under force[J]. Sci Rep-Uk. 2016,6. Camacho KM, Kumar S, Menegatti S, Vogus DR, Anselmo AC, Mitragotri S. Synergistic antitumor activity of camptothecin-doxorubicin combinations and their conjugates with hyaluronic acid[J]. J Control Release. 2015,210:198-207. Zhao YQ, Zhang T, Duan SF, Davies NM, Forrest ML. CD44-tropic polymeric nanocarrier for breast cancer targeted rapamycin chemotherapy[J]. Nanomed-Nanotechnol. 2014,10(6):1221-30. Oh SS, Lee BF, Leibfarth FA, Eisenstein M, Robb MJ, Lynd NA, et al. Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells[J]. Journal of the American Chemical Society. 2014,136(42):15010-5. Li MQ, Tang ZH, Lin J, Zhang Y, Lv SX, Song WT, et al. Synergistic Antitumor Effects of Doxorubicin-Loaded Carboxymethyl Cellulose Nanoparticle in Combination with Endostar for Effective Treatment of Non-Small-Cell Lung Cancer[J]. Advanced Healthcare Materials. 2014,3(11):1877-88. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3972887","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274827517,"identity":"36cc8c4f-7ace-4a09-8b0a-e6c2f911ba64","order_by":0,"name":"Fengjun Liu","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Fengjun","middleName":"","lastName":"Liu","suffix":""},{"id":274827518,"identity":"4a921c1d-6fe2-4311-aa09-3b93f397e46a","order_by":1,"name":"Zhiyang Xu","email":"data:image/png;base64,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","orcid":"","institution":"Shanghai Medical College of Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Zhiyang","middleName":"","lastName":"Xu","suffix":""},{"id":274827519,"identity":"b1465638-2092-4d68-bbdd-bbd859033087","order_by":2,"name":"Xiaochao Jia","email":"","orcid":"","institution":"Jinshan Hospital of Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaochao","middleName":"","lastName":"Jia","suffix":""},{"id":274827520,"identity":"1cd56ff0-844a-47bd-bad8-6298fe2dcf52","order_by":3,"name":"Yidan Tang","email":"","orcid":"","institution":"Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yidan","middleName":"","lastName":"Tang","suffix":""},{"id":274827521,"identity":"6e36f800-457d-42e0-a313-c89084ef10ee","order_by":4,"name":"Mingsheng Chen","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Mingsheng","middleName":"","lastName":"Chen","suffix":""},{"id":274827522,"identity":"54da5753-7c0c-443e-9e16-f6234013ddc5","order_by":5,"name":"Chuan Chen","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Chuan","middleName":"","lastName":"Chen","suffix":""},{"id":274827523,"identity":"e5a53c9f-559e-4f79-b612-eb260d6b28bb","order_by":6,"name":"Fang Fang","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Fang","suffix":""},{"id":274827524,"identity":"f76d0332-42cd-4149-89e7-7ddca3982b45","order_by":7,"name":"Xiudong Shi","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Xiudong","middleName":"","lastName":"Shi","suffix":""},{"id":274827525,"identity":"7f158042-d0da-46cb-a17d-6da57b4a7a7d","order_by":8,"name":"Tianyou Chen","email":"","orcid":"","institution":"Shanghai Traditional Chinese Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianyou","middleName":"","lastName":"Chen","suffix":""},{"id":274827526,"identity":"add52202-244f-4282-8ba4-a5968263b581","order_by":9,"name":"Yuxin Shi","email":"","orcid":"","institution":"Shanghai Public Health Clinical Center","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2024-02-20 13:32:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3972887/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3972887/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51621419,"identity":"fa5e9193-37ec-4017-baf0-03e22dc7e6f1","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":993313,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Flow cytometry showed that 99% of pancreatic cancer cells expressed CD44. (B, C) Pancreatic cancer cells and tissues had high CD44 expression (brown).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/23d8a7219ea63edcfbd07aee.png"},{"id":51621422,"identity":"bda50de4-16bb-417d-9b07-b8ee58fa187d","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":577242,"visible":true,"origin":"","legend":"\u003cp\u003e(A-D) MITO and MITO@HA characterization. (E) Different concentrations of MITO and MITO@HA changed cell survival. (F) Treatment with MITO@HA at different concentrations and for different durations had statistically significant effects on the survival and inhibition rates of pancreatic cancer cells .\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/408a9520bd557c04d6d0949a.png"},{"id":51621424,"identity":"76bd35e9-ead3-49b5-931f-2f3bda6ef212","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1291414,"visible":true,"origin":"","legend":"\u003cp\u003e(A-D) Comparison of cell cycle changes. Microscopy revealed increased apoptosis, decreased cell migration and invasion, and decreased cell clones of PANC-1 cells in the control, MTIO@HA and MITO groups. The cell functions in each group were significantly different.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/1be1b07b106432173ab73ba4.png"},{"id":51621421,"identity":"e80e165b-0ae3-46c7-a5ef-d3d63a259bc0","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":811746,"visible":true,"origin":"","legend":"\u003cp\u003eCompared with the control group, (A) the tumor growth curve of pancreatic cancer animals was slowed down. (B) the tumor weight was lighter after 26 days of tumor growth. (P＜0.0001).(C) HE staining revealed that MITO@HA caused massive tumor necrosis. There was a statistically significant difference between each group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/a7f9c7fcc37a52a5a58c2c5e.png"},{"id":51621418,"identity":"25f67f0f-8d53-4429-9416-a7c4fb1e43e0","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186167,"visible":true,"origin":"","legend":"\u003cp\u003eCompared to those of the control group, the blood parameters of the MITO@HA group were normal, the liver and kidney function indices were normal, and the myocardial enzyme spectrum was normal.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/b28fb94727dbf1cc8f6fc9f0.png"},{"id":73845635,"identity":"0b4a69e1-3d70-495a-b024-c0fcc8afea8b","added_by":"auto","created_at":"2025-01-15 09:02:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5370920,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/47e4a01b-5c16-4749-a162-99a3603bb39b.pdf"},{"id":51621420,"identity":"725f6c52-bb51-4e26-a7da-31a9ace41ac4","added_by":"auto","created_at":"2024-02-26 05:56:39","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":202602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3972887/v1/0cf4dc789ab05d3e3727101d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Drug-loaded MITO@HA nanodrugs for evaluating the efficacy of targeted therapy for pancreatic cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePancreatic cancer is one of the most lethal digestive system malignancies and is both highly aggressive and metastatic, accounting for more than 200,000 deaths per year\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Due to the lack of typical early-stage clinical manifestations, diagnosis is difficult, and 80% of patients are diagnosed at an advanced stage, limiting the efficacy of surgery\u003csup\u003e4\u003c/sup\u003e. Therefore, chemotherapy plays a very important role in treating pancreatic cancer. Gemcitabine, cisplatin, and paclitaxel are the major current therapeutic options for pancreatic cancer, but their use is limited by strong toxicity and low therapeutic efficacy.\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e In addition, a number of potent small molecules targeting PDK are important milestones in the history of cancer research, including for example, dichloroacetic acid (DCA) and its derivatives, dihydroxyphenylpyrazole M77976, hordenine, quercetin, and the sulfone AZD7545 (which showed potent antiproliferative activity in different cancer cell lines and mouse models). Unfortunately, because of their low potency or poor selectivity, none of these small molecules have succeeded in passing clinical trials\u003csup\u003e8\u0026ndash;10\u003c/sup\u003e. Clinical trial data are still lacking, and additional studies are urgently needed to verify the relevant effects and to develop more personalized treatment approaches.\u003c/p\u003e \u003cp\u003eTo improve the current situation, researchers have developed a variety of polymer nanoparticles as drug carriers, generally approximately 10\u0026thinsp;~\u0026thinsp;200 nm in diameter, which use their various advantages, including targeting and stimulating response release, to effectively improve the therapeutic efficacy against tumors\u003csup\u003e11\u0026ndash;14\u003c/sup\u003e. Improving the survival rate of pancreatic cancer patients has always been a difficult problem for domestic and foreign scholars. Due to the poor drainage of lymph fluid in pancreatic cancer patients, which results in high permeability and long retention and targeting effects, nanodrugs of suitable size can enter tumors through relatively small blood vessels with relatively tight intercellular spaces, stay in tumors for several days, both passively and actively accumulate at relatively high concentrations \u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. The study of polymer nanoparticles for the treatment of pancreatic cancer is currently uncommon and is still in the exploratory research stage. Hyaluronic acid (HA) is an acidic mucopolysaccharide that is the main component of the extracellular matrix, and its structure is a repeating unit composed of D-glucuronic acid and N-acetylglucosamine, which are water soluble, biocompatible, and biodegradable\u003csup\u003e18\u0026ndash;24\u003c/sup\u003e. More importantly, hyaluronic acid also targets the CD44 receptor\u003csup\u003e25\u003c/sup\u003e, which can accumulate and aggregate in a variety of malignant tumor tissues overexpressing the CD44 receptor, increasing the concentration of local drugs; therefore, it has been widely studied and used in the field of nanodrug delivery. Mitoxantrone hydrochloride (MITO), which binds to DNA molecules, inhibits nucleic acid synthesis, and causes cell death, is a nonspecific drug used for the cell cycle\u003csup\u003e26\u0026ndash;27\u003c/sup\u003e. Some studies have shown that mitoxantrone has serious side effects, such as myelosuppression and cardiotoxicity, which seriously impede the use of its conventional formulations in the clinic\u003csup\u003e28\u0026ndash;30\u003c/sup\u003e. In recent years, to enhance MITO\u0026rsquo;s antitumor effects and reduce its toxic side effects, MITO research has mainly focused on targeted drug delivery systems. Based on previous research\u003csup\u003e31\u003c/sup\u003e, this paper constructed a MITO@HA nanodrug synthesized with sodium hyaluronate as a targeted drug carrier and wrapped it with mitoxantrone hydrochloride to evaluate the antitumor effect on pancreatic cancer (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eCell culture reagents: fetal bovine serum (HyClone, Cat.No.SH30087.01), RPMI-1640 medium (HyClone, Cat.No.SH30809.18), DMEM-high sugar medium (HyClone, cat.No.SH30019.01), penicillin (HyClone, Cat.No. SH30010), PBS potassium phosphate buffer (HyClone, Cat. No. SH30256.01B), an Annexin V-FITC apoptosis assay kit (Keygen, No. KGA106), a cell cycle assay kit (Keygen, No. KGA511), a Transwell cell culture plate (BD, No. REF353097), a Matrigel gel (BD, No. 356234), and mitoxantrone hydrochloride (MCE, No. HY-13502A) were used.\u003c/p\u003e \u003cp\u003eA microplate reader (Thermo Fisher Scientific No. Multiscan MK3) and an ELISPOT (AID iSpot system) were used. A Suzhou Antai Clean Workbench (SW-CJ-IFD), a low-speed centrifuge (Zhongjia, SC3614), an inverted light microscope (OLYMPUS CKX41, U-CTR30-2), a cell thermostatic incubator (Thermo Scientific, HERACELL150i), an inverted fluorescence microscope (Leica, No. DMI6000B), flow cytometry (BD Calibur), a camera system (Guangzhou Mingmei Technology Co., Ltd., No. MC30), and software analysis (Java, No. ImageJ 1.44) were used. SPF-grade BALB/C-NU nude mice were purchased from Guangdong Pharma Biotechnology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Expression of CD44 in pancreatic cancer cells\u003c/h2\u003e \u003cp\u003eFirst, 0.5 ml of 0.25% pancreatic enzyme without EDTA was added to PANC-1 pancreatic cancer cells and incubated until cells were observed under a microscope to begin to detach from the culture plate wall. The cells were gently resuspended in medium from step 1 or prechilled with 1\u0026times;binding buffer to a density of approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. Then, 0.5 mL of the cell suspension from the cell culture plate (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) was transferred into a clean centrifuge tube. After adding FITC mice transferrin to the anti-human CD44 antibody and incubating it in the dark for 30 min, the cells were centrifuged with PBS 3 times, immediately analyzed via flow cytometry.\u003c/p\u003e \u003cp\u003eThe sterilized 20 mm coverslips were placed in a 90 mm dish, the cells were seeded in the dish at a density of 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e/ml PANC-1 pancreatic cancer cells for cell crawling, and immune cell and histochemical staining was performed 5 days later. The cells were washed with PBS three times for 2 min each And then fixed in 4% paraformaldehyde for 15 min (cold methanol:acetone 1:1) and air-dried for 5 min. The cells were then washed three times with PBS for 2 min each. The cells were incubated with 0.5% Triton X-100 (with DPBS) once for 20 minutes. The cells were washed with PBS three times for 2 min each. The mixture was incubated with 3% H2O2 for 15 min. The cells were washed with PBS three times for 2 min each. Samples were then incubated with blocking serum (5% normal secondary antibody serum DPBS solution) for 20 min, followed by primary antibody incubation (PBS with titers 1:200, wet box) at 40\u0026deg;C overnight or at 37V for 60 min. The negative control was cleaned with primary antibody-derived serum and PBS three times for 5 min each. The secondary antibody working solution was incubated with PV6001 (wet box) at 37\u0026deg;C for 30 min. The specimens were washed 5 times with PBS for 2 min each. DAB was used for color development (protected from light, observed to brown under the microscope) for approximately 10\u0026thinsp;~\u0026thinsp;15 minutes. Finally, the samples were washed once in distilled water for 5 min, counterstained with hematoxylin for 10 min, and then washed once again in distilled water for 5 min. Afterwards, the samples were sealed and all photos collected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Preparation of the nanoprobe of MITO@HA\u003c/h2\u003e \u003cp\u003e300 mg of sodium hyaluronate was weighed with a balance, dissolved in 50 ml of deionized water, and stirred for 10 min to dissolve it thoroughly. Then, 30 mg of mitoxantrone hydrochloride was weighed, dissolved in 5 ml of deionized water until fully dissolved, added dropwise to sodium hyaluronate solution (3\u0026ndash;4 drops/min), and stirred overnight. The extra free drug was removed by dialysis (MWCO 3500 Da) for 24 hours, after which the solution was lyophilized to obtain the MITO@HA powder. All the procedures were performed in the dark. The particle size, drug load (DLC), and drug carrying rate (DLE) of MITO@HA were determined via ultraviolet spectroscopy and inductively coupled plasma atomic emission spectrometry. DLC and DLE are calculated using the following formulas:\u003c/p\u003e \u003cp\u003eDLC(wt.%)= (mass of nanoparticle-loaded drug) / (total amount of drug - mass of nanoparticles)\u0026times;100%\u003c/p\u003e \u003cp\u003eDLE(wt.%)= (mass of nanoparticle-loaded drug) / (mass of total drug input) \u0026times;100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell culture and grouping\u003c/h2\u003e \u003cp\u003ePANC-1 pancreatic cancer cell lines were cultured in 1640 culture medium supplemented with 10% fetal bovine serum and 1% biclonal antibody and subcultured in an incubator with 5% CO2 saturated humidity and 37\u0026deg;C. The cells were subpassaged, cultured in 12-well plates and divided into control groups, MITO groups, and MITO@HA groups (6 complex wells per group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eThe CCK-8 method was used to incubate pancreatic cancer cells for 1 d in combination with MITO@HA (0 \u0026micro;g/ml, 1 \u0026micro;g/ml, 2.5 \u0026micro;g/mL, 5 \u0026micro;g/mL, 20 \u0026micro;g/mL, 50 \u0026micro;g/mL). Additionally, seven different concentrations of 0 \u0026micro;mol/L, 0.1 \u0026micro;mol/L, 0.25 \u0026micro;mol/L, 0.5 \u0026micro;mol/L, 1 \u0026micro;mol/L, 2 \u0026micro;mol/L and 4 \u0026micro;mol/L were added to the control group, MITO group and MITO@HA groups, respectively. After digesting each group of cells, the cell count was adjusted to 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, divided into 96-well plates, and 100 \u0026micro;L wells, that is, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well. Adherent cells were allowed to adhere, after which the cells were collected at each time point for detection. Cells (0 h, 24 h, 48 h, 72 h) were collected at various time points and added to CCK-8 solution (Keygen Bio, Cat.No. KGA317), the scale is 1/10. Briefly, 100 \u0026micro;l of culture solution was added to 10 \u0026micro;l of detection solution. After 4 h of incubation, the microplate reader was used to read the plate, and the OD450 data were obtained via the CCK-8 assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell cycle\u003c/h2\u003e \u003cp\u003eThe cells in the control group, MITO group, and MITO@HA group were incubated with 0.5 \u0026micro;mol/L PBS, MITO, or MITO@HA for a total of 2 days and incubated with 0.25% pancreatic enzyme without EDTA until microscopic observation that the cells had begun to detach from the culture plate wall. The cells were collected by centrifugation, the supernatant was discarded, the cells were washed twice with precooled PBS, precooled 70% ethanol was added, and the cells were fixed overnight at 4 ℃ or fixed for a long time at -20 ℃. The cells were centrifuged and collected, washed with 1 mL of PBS once, 500 \u0026micro;l of PBS containing 50 \u0026micro;g/mL propidium bromide (PI), 100 \u0026micro;g/mL LRNaseA, and 0.2% Triton X-100 was added, and the mixture was incubated in the dark at 4\u0026deg;C for 30 minutes. Using standard procedures, flow cytometry was used to detect and count 20000 to 30000 cells. The results were analyzed using the cell cycle fitting software ModFit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Cell apoptosis\u003c/h2\u003e \u003cp\u003eThe cells in the Control, MITO, and MITO@HA groups were incubated for 2 days in MITO@HA supplemented with 0.5 ml of 0.25% pancreatic enzyme without EDTA until the cells were observed to detach from the plate wall via microscopy. The cells were gently resuspended in medium from step 1 or prechilled with 1\u0026times; binding buffer to a density of approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL. 0.5 mL of the cell suspension was then transferred from the cell culture plate (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells) into a clean centrifuge tube. Then, 1.25 \u0026micro;L of Annexin V-FITC was added. The mixture was protected from light for 15 min at room temperature (18\u0026ndash;24\u0026deg;C). The mixture was centrifuged at room temperature (1,000\u0026times;g) for 5 min to remove the supernatant. The cells were gently resuspended in 0.5 ml of prechilled 1\u0026times;binding buffer. 10 \u0026micro;L of propidium iodide was added to the sample solution. The samples were stored on ice protected from light. The cells were then detected via flow cytometry for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Cell cloning\u003c/h2\u003e \u003cp\u003eThe cells in the control group, MITO group, and MITO@HA group were incubated with 0.5 \u0026micro;mol/L PBS, MITO, and MITO@HA for a total of 2 days, cells from the different groups were collected via trypsinization, and the cells were counted in 1 ml of medium. Pipette into a single-cell suspension, count the cells, dilute the cells into 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/ml, seed in a 6-well plate, and pipette 300 \u0026micro;l seeded to 300 cells/well. After inoculation, each well was supplemented with medium to 2000 \u0026micro;l/well. The plate was shaken back and forth horizontally to ensure that the cells were evenly distributed in the wells. Every day, when a single cell grew into a clump-like clone, generally after 7 days of culture, each well of medium was removed, the cells were rinsed with PBS or normal saline 2 times, and each well was supplemented with 200 \u0026micro;l of crystal violet staining solution to fully cover the bottom of the well. After 20 min, the 96-well plate was rinsed with tap water, the cells were allowed to proliferate, and the flow of water was decreased. After rinsing, the number of colonies was calculated. Analyses, scans and photographs were taken using an enzyme-linked spot image automatic analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Cell migration and invasion\u003c/h2\u003e \u003cp\u003eThe cell culture plate Control group, MITO group, and MITO@HA group were each incubated for 2 days with 0.5 \u0026micro;mol/L PBS, MITO, MITO@HA, respectively, cells from the treatment group were collected, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were counted, resuspended with 100 \u0026micro;l serum-free medium, added to the upper chamber of the Transwell cell culture plate, and 600 \u0026micro;l complete medium was added to the lower chamber. The cell culture plates were CO\u003csub\u003e2\u003c/sub\u003e incubated at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e for 12\u0026ndash;48 h, taken out the chamber, and the cells in the upper chamber were wiped off with a cotton swab. Cells were then fixed in 4% paraformaldehyde for 20 min, wash with PBS once, were crystal violet stained for 10 min, and then washed with PBS again. Afterwards, the cells were observed under the microscope to determine whether the cells passed through the small wells. If the cells passed through other experimental groups, images were taken for statistical analysis. Upper ventricular lower surface cells stained with crystal violet were eluted with 33% acetic acid, after which the OD570 was determined via a microplate reader. The Matrigel was dissolved overnight at 4\u0026deg;C, diluted with prechilled serum-free medium at a volume ratio of 1:3, 40 \u0026micro;l was added to the prechilled Transwell chamber, and the mixture was incubated at 37\u0026deg;C for 2 h to solidify the Matrigel. The excess liquid in the chamber was aspirated, 100 \u0026micro;l and 600 \u0026micro;l of serum-free medium were added to the upper chamber and lower chamber, respectively, and the cells were equilibrated at 37\u0026deg;C overnight. One day after the drugs were added, 1\u0026times;105 cells were counted, the cells were resuspended in 100 \u0026micro;l of serum-free medium, the Transwell chamber was added to the upper chamber, and 600 \u0026micro;l of complete medium was added to the lower chamber. After incubation at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 and 48 hours, the cells were taken out the chamber, the cells in the upper chamber were wiped off with a cotton swab, and then fixed 4% paraformaldehyde for 15 min. Afterwards, they were washed once with PBS, stained with crystal violet for 10 min, washed again with PBS. If the cells pass through the small wells and through other experimental groups, pictures for statistics were. Upper ventricular lower surface cells stained with crystal violet were eluted with 33% acetic acid, after which the OD570 was determined via a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Tumor Inhibition Studies\u003c/h2\u003e \u003cp\u003eFive-week-old male nude mice received 0.1 ml (containing 5\u0026times;107 PANC-1 cell suspension in nude mice for subcutaneous tumorization). The tumor-bearing nude mice were randomly divided into 3 groups: the control group; the MITO group, and the MITO@HA group (n\u0026thinsp;=\u0026thinsp;3 per group). The tumors of tumor-bearing nude mice were allowed to grow to 3-4mm, and the mice all received intravenous injection of PBS, MITO, or MITO@HA at a 10 mg/kg equivalent dose, which amounted to an injection dose of 100 \u0026micro;l. The mice were injected every three days for a total of 3 times. Mouse tumor volume was measured every 3 days. On Day 26 after tumoring, the mice were sacrificed by inhalation of carbon dioxide, after which the tumors were harvested and weighed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Toxicity assessment\u003c/h2\u003e \u003cp\u003ePancreatic cancer tumor-bearing nude mice were divided into three groups: control group, MITO group and MITO@HA group (n\u0026thinsp;=\u0026thinsp;3 per group). Tumor-bearing nude mice were intravenously injected with PBS, MITO, or MITO@HA at a 10 mg/kg equivalent dose, and 100 \u0026micro;l was injected once every three days for a total of 3 injections. On the 26th day after tumor seeding, the mice were sacrificed by inhalation of carbon dioxide, and eye blood was collected to detect blood parameters, liver and kidney function, and cardiac enzymes for safety assessment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical results of the measurement data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), and the statistical analysis and graphing software used SPSS and GraphPad Prism. After satisfying the normality distribution and homogeneity of variance tests, the two groups of data were compared via an independent t test, and the comparisons between multiple groups of data were performed via one-way ANOVA. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates that the difference is statistically significant; * indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, **** indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and **** indicates P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cem\u003e3.1. Expression of CD44 in pancreatic cancer cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn recent years, nanotechnology has become one of the most important technologies at present, playing an important role in the rapid development of several research fields, especially in the field of precision cancer treatment. HA has good biocompatibility and CD44 receptor targeting\u003csup\u003e32-33\u003c/sup\u003e, which can accumulate and aggregate in PANC-1 pancreatic cancer tumor tissues overexpressed by CD44 receptor, target the release of chemotherapy drugs, and improve anti-tumor efficiency. Flow cytometry to determine CD44 expression levels in PANC-1 pancreatic cancer cells. The results (Figure 1A) showed that 99.9% of pancreatic cancer cells significantly overexpressed CD44.Pancreatic cancer cells (Figure 1B) and tissue immunohistochemistry(Figure 1C) \u0026nbsp;showed high expression of CD44.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2. Constructing MITO@HA\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.1 Characterization of MITO@HA\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we successfully created an MITO@HA-targeted nanomedicine through the hydrophilic interaction of HA\u0026nbsp;with\u0026nbsp;MITO, and we evaluated its therapeutic efficacy on pancreatic cancer cells in vitro and\u0026nbsp;on pancreatic cancer animals in vivo via tail vein injection in nude mice to investigate its targeted inhibitory effect on pancreatic cancer.\u0026nbsp;Compared with traditional chemical drugs, polymer nanoparticle-loaded antitumor drugs offer advantages: (1) targeted therapy\u003csup\u003e34\u003c/sup\u003e and (2) enhanced tumor accumulation\u003csup\u003e35\u003c/sup\u003e. MITO@HA nanodrugs can enhance the antitumor efficacy and reduce toxic side effects of these drugs by actively targeting CD44Rs and enhancing the permeability and retention (EPR) effect on the surface of pancreatic cancer tumors.\u003c/p\u003e\n\u003cp\u003eFirst, solutions at different concentrations (10\u0026nbsp;\u0026mu;g/ml, 15\u0026nbsp;\u0026mu;g/ml, 25\u0026nbsp;\u0026mu;g/ml, 35\u0026nbsp;\u0026mu;g/ml, 50\u0026nbsp;\u0026mu;g/ml, 75\u0026nbsp;\u0026mu;g/ml) were prepared with a mitoxantrone hydrochloride standard aqueous solution, and the ultraviolet absorption spectrum was determined via an ultraviolet spectrophotometer, with 610 nm as the maximum absorption peak. The linear relationship between the concentration of the drug and the absorbance was determined as a standard curve (Figure 2A and Figure 2B). By measuring the absorbance of MITO@HA at 610 nm, the encapsulation rate and drug loading were calculated to be 45.82\u0026plusmn;1.72% and 4.15\u0026plusmn;0.82%, respectively (Figure 2C). The authors demonstrated the successful self-assembly of nanoparticles between hyaluronic acid and mitoxantrone through electrostatic interactions. This study provides a basis for subsequent active targeted therapy for pancreatic cancer. Next, the morphological characteristics and particle size distributions of the nanoions were analyzed. MITO@HA The average hydrated particle size measured by a dynamic light scattering nanolaser was 51.4\u0026nbsp;\u0026plusmn;\u0026nbsp;2.3 nm, and the particle size in the TEM image was consistent with the particle size measured by particle size analysis. Moreover, the surface of MITO@HA had high electrostatic stability (-26.1\u0026nbsp;\u0026plusmn;\u0026nbsp;3.2 mV) (Figure 2D).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2.2 Screening of the MITO@HA concentration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter treating pancreatic cancer cells with 1 \u0026mu;g/ml, 2.5 \u0026mu;g/mL, 5 \u0026mu;g/mL, 20 \u0026mu;g/mL, and 50 \u0026mu;g/mL MITO, as determined by the CCK-8 method, it was found that the concentration of MITO@HA significantly inhibited the proliferation of cells in the MITO group (Figure 2E). When the concentration was increased to \u0026mu;mol/L, the efficacy and toxicity of seven different concentrations of MITO@HA were evaluated in pancreatic cancer cells after 0, 1, 2 and 3 days. MITO@HA was incubated with pancreatic cancer cell precursors at a concentration of 0.5 \u0026mu;mol/L for 2 days at the most appropriate concentration and time (Figure 2F).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3\u0026nbsp;\u003c/em\u003eIn vitro\u003cem\u003e\u0026nbsp;study\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;the present\u0026nbsp;study,\u0026nbsp;inhibition of pancreatic cancer cells\u0026nbsp;by\u0026nbsp;MITO@HA\u0026nbsp;for 2 days was most suitable for\u0026nbsp;treating\u0026nbsp;pancreatic cancer cells at a concentration of 0.5 \u0026mu;mol/L. Compared with\u0026nbsp;those in the MITO group,\u0026nbsp;the cells in the MITO@HA\u0026nbsp;group exhibited\u0026nbsp;cell cycle\u0026nbsp;arrest at the G0/G1 phase, increased apoptosis, decreased cell replication, cell migration and invasion inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3.1 Cell cycle\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cell cycle results (Figure 3A) showed that the average percentages for each group were 24.91\u0026plusmn;1.42%、39.37\u0026plusmn;1.27%和42.67\u0026plusmn;0.944%, respectively, in the Control group, MITO and MITO@HA groups, indicating that nanoparticles affected the cycling activity of PANC-1 pancreatic cancer cells, resulting in most cells in G0/G1 phase blockade (P\u0026lt;0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3.2 Cell apoptosis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptosis assay results revealed that (Figure 3B) the average percentages of the sum of the total apoptosis rates (early apoptosis rate Q4 and late apoptosis rate Q2) in the control, MITO, and MITO@HA groups were 3.01\u0026plusmn;0.12%, 5.30\u0026plusmn;0.49% and 7.28\u0026plusmn;0.59%, respectively (P\u0026lt;0.0001). MITO@HA had the greatest impact on the apoptosis of PANC-1 pancreatic cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3.3 Cell migration and invasion\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe results of cell migration and invasion are shown in Figure 3C. The average migration and invasion rate of PANC-1 cells in the control group was 280.75\u0026plusmn;18.51%, which is consistent with the rapid metastasis of pancreatic cancer. The mobility rates of the patients in the MITO@HA and MITO groups were significantly lower than those in the control group (P\u0026lt;0.0001), with 154.50\u0026plusmn;9.02% and 145.63\u0026plusmn;7.15%, respectively. Moreover, there was no statistically significant difference between the MITO group and the MITO@HA group (P=0.3539). Similarly, cell invasion was significantly lower in the MITO@HA and MTO groups than in the control group (P\u0026lt;0.0001). The above results showed that MTO has a significant inhibitory effect on tumor growth. In addition, hyaluronic acid (HA) targets the CD44 receptor in pancreatic cancer, and HA-coated MTO can also tightly adhere to the CD44 receptor and target it to inhibit the migration and invasion of tumor cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3.4 Cell cloning\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cell cloning results showed that (Figure 3 D) for the control, MITO, and MITO@HA groups, the average percentages of cell clones in each group were 100.00\u0026plusmn;3.56%, 47.02\u0026plusmn;9.53%, and 45.02\u0026plusmn;7.38%, respectively. The number of clones of PANC-1 pancreatic cancer cells in the MITO@HA group was the smallest and was significantly lower than that in the control and MITO groups.\u003c/p\u003e\n\u003cp\u003eTherefore, we believe that hyaluronic acid (HA) targets the CD44 receptor in pancreatic cancer and plays the most important role in killing pancreatic cancer cells. In addition, the constructed MITO@HA nanodrugs target and concentrate in the plasma of pancreatic cancer cells and release MITO, resulting in significant blockade of the G0/G1 cell cycle in pancreatic cancer, promotion of tumor cell apoptosis, significant reduction of cell cloning, and inhibition of cell migration and invasion.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4.\u0026nbsp;\u003c/em\u003eIn vivo\u003cem\u003e\u0026nbsp;study\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe results of animal experiments showed that MITO@HA nanodrugs had a significant inhibitory effect on pancreatic tumors. A tumor growth curve was generated for the nude mice with pancreatic cancer (Figure 4A). The growth of the tumors in the control group was the fastest, and the growth of the tumors in the MITO@HA group was slower than that in the control group, indicating that the tumor inhibition effect of the former was significant. The average tumor mass of the nude mouse tumors in each group (Figure 4B) was 0.846\u0026plusmn;0.016 g in the control group and 0.790\u0026plusmn;0.0346 g in the MITO@HA group, and the average tumor mass in the MITO@HA group was 0.455\u0026plusmn;0.529 g (P\u0026lt;0.0001). To further validate the antitumor efficacy of the targeting liposomes, tumor tissue was fixed, sectioned, and HE stained (Figure 4C). Compared with those in the control and MITO groups, the tumor cells in the MITO@HA group exhibited obvious large areas of liquefied necrosis, whereas the control group exhibited no obvious necrosis. The above results showed that MITO@HA had an obvious inhibitory effect on tumors and had good therapeutic effects on tumors. The reason for this difference is that MITO@HA can allow more drugs to reach tumor tissue through EPR effects, and HA can fuse with cell membranes to swallow lipids into cells, increasing tumor cell uptake. In vivo antitumor experiments further proved that surface-modified HA can further promote the accumulation and uptake of MITO@HA at the tumor site through passive and active targeting to better suppress tumor growth.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.5.\u0026nbsp;\u003c/em\u003eIn vivo\u003cem\u003e\u0026nbsp;toxicity assessment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe biochemical indices of the nude mice in each preparation group are shown in Figure 5. Important parameters such as white blood cells, red blood cells, PLT, ALT, AST, TBIL, ALB, CK, CK-MB, LDH, LDB1, URES, CREA, and UA in the MITO@HA group were normal, indicating that the MITO@HA treatment had good safety.\u003c/p\u003e\n\u003cp\u003eIn this study, the biosafety of MITO@HA nanoparticles was further confirmed by blood biochemical indices and enhanced antitumor effects compared to those of the MTIO group. In summary, MITO@HA nanoparticles targeting the CD44 receptor can effectively enhance the therapeutic efficacy against pancreatic cancer tumors, and the safety of these nanoparticles is reliable, suggesting the possibility of clinically targeted treatment for pancreatic cancer.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent years, nanotechnology has become one of the most important technologies, playing a crucial role in the rapid development of various research fields, especially in the field of precision cancer treatment. Hyaluronic Acid (HA) exhibits good biocompatibility and CD44 receptor targeting specificity\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e.Accumulating in pancreatic cancer tissues with overexpressed CD44 receptors, targeted release of chemotherapy drugs can enhance the anti-tumor efficiency. In this study, hyaluronic acid (HA) was chosen as the raw material, and through hydrophilic and hydrophobic interactions, MITO was loaded to synthesize hyaluronic acid MITO@HA nano-drugs. The study aimed to verify and explore its targeted inhibitory effect on pancreatic cancer by intravenous injection of MITO@HA in nude mice with pancreatic cancer.。Compared to traditional chemotherapy drugs, anti-tumor drugs loaded onto polymer nanoparticles have advantages: ①Targeted therapy\u003csup\u003e14\u003c/sup\u003e,②Enhanced tumor accumulation\u003csup\u003e15\u003c/sup\u003e.The MITO@HA nano-drug can enhance its anti-tumor efficacy through active targeting to the surface CD44 receptors of pancreatic cancer tumors and the enhanced permeability and retention (EPR) effect, thereby reducing toxic side effects. In this study, pancreatic cancer cell experiments showed that the toxicity and safety of MITO@HA were most suitable when treating pancreatic cancer cells for 2 days at a concentration of 0.5 \u0026micro;mol/L. Compared to the MITO group, MITO@HA exhibited G0/G1 phase arrest in the cell cycle, increased apoptosis, reduced cell cloning, and inhibited cell migration and invasion.The results of animal experiments show that the MITO@HA nano-drug has a significant inhibitory effect on pancreatic tumors. This study further confirmed the biocompatibility of MITO@HA nanoparticles through the detection of blood biochemical indicators, demonstrating its ability to effectively reduce damage to animals caused by chemotherapy drugs. Additionally, MITO@HA enhances the anti-tumor effect of chemotherapy drugs.In summary, MITO@HA nanoparticles, with CD44 receptor targeting specificity, can effectively reduce systemic drug toxicity while enhancing the therapeutic effect on pancreatic cancer tumors. This offers a potential avenue for clinical targeted treatment of pancreatic cancer.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eBased on the characteristics of CD44 receptors, which are highly expressed in pancreatic cancer tumor cells, MITO@HA, which can target the delivery of antitumor drugs, was prepared by using HA to specifically bind to CD44 receptors. MITO@HA was characterized, and the results showed that MITO@HA had a uniform particle size distribution. The results of cell-based experiments showed that MITO@HA-free MITO could significantly increase uptake in pancreatic cancer cells; inhibit tumor cell proliferation, apoptosis, migration, and invasion; and cause cell cycle arrest at the G0/G1 phase. The results of in vivo antitumor experiments proved that MITO@HA can effectively inhibit the growth of tumors and have good antitumor effects, and the animal biochemical indices indicated that they were normal. In summary, HA can target and modify nanodrugs, thereby increasing the targeting efficiency of nanodrugs and improving the antitumor efficiency and safety of MITO@HA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions:\u003c/strong\u003eFengjun Liu and Yu Xinshi conceived and designed the study.\u0026nbsp;Tianyou Chen\u0026nbsp;wrote the manuscript.\u0026nbsp;Zhiyang Xu and rewriting the manuscript.\u0026nbsp;Shi Xiudong, Zhiyang Xu, Jia Xiaochao, Yidan Tang, Mingsheng Chen, Chuan Chen, and Fang Fang\u0026nbsp;and interpreted the data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe research was supported by the National Natural Science Foundation of China (Grant 82172029, 82302265), the Shanghai Municipal Health Commission (Grant 202140084), Shanghai Pujiang Program (Grant 21PJD062), the Shanghai Municipal Health Commission, Key discipline construction project of Shanghai Three-year Action Plan for Public Health System Construction (Grant GWV-10.1-XK09)，the National Natural Science Foundation of China (Grant 82302265) , and Shanghai Sailing Program (Grant 21YF1436600). The funder had no role in study design, data collection, analysis, the decision to publish, or the preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset presented in the study is available on request from the First author (Fengjun Liu) during submission or after publication. The data are not publicly available due to privacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShanghai Public Health Clinical Center Laboratory Animal Welfare \u0026amp; Ethics committee is the animal ethics committee that reviewed our study.\u003c/p\u003e\n\u003cp\u003eThe relevant work of the Shanghai Public Health Ethics Committee follows the \"Regulations on the Management of Experimental Animals\" issued and implemented by the National Science and Technology Commission, and complies with relevant Chinese laws and regulations\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eVincent A, Herman J, Schulick R, Hruban R H, Goggins M,et al.Pancreatic cancer［J］.Lancet. 2011,378(9791):607-620.\u003c/li\u003e\n \u003cli\u003eWael R,Abd-Elgaliel,Ching-Hsuan Tung,et al.Selective detection of Cathepsin E proteolytic activity[J]. 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Synergistic antitumor activity of camptothecin-doxorubicin combinations and their conjugates with hyaluronic acid[J]. J Control Release. 2015,210:198-207.\u003c/li\u003e\n \u003cli\u003eZhao YQ, Zhang T, Duan SF, Davies NM, Forrest ML. CD44-tropic polymeric nanocarrier for breast cancer targeted rapamycin chemotherapy[J]. Nanomed-Nanotechnol. 2014,10(6):1221-30.\u003c/li\u003e\n \u003cli\u003eOh SS, Lee BF, Leibfarth FA, Eisenstein M, Robb MJ, Lynd NA, et al. Synthetic aptamer-polymer hybrid constructs for programmed drug delivery into specific target cells[J]. Journal of the American Chemical Society. 2014,136(42):15010-5.\u003c/li\u003e\n \u003cli\u003eLi MQ, Tang ZH, Lin J, Zhang Y, Lv SX, Song WT, et al. Synergistic Antitumor Effects of Doxorubicin-Loaded Carboxymethyl Cellulose Nanoparticle in Combination with Endostar for Effective Treatment of Non-Small-Cell Lung Cancer[J]. Advanced Healthcare Materials. 2014,3(11):1877-88.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MITO@HA, CD44, Nanomedicine carriers, Pancreatic cancer, Antitumor effect, High security","lastPublishedDoi":"10.21203/rs.3.rs-3972887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe purpose of this study was to evaluate the efficiency of mitoxantrone hydrochloride (MITO@HA) as a targeted pancreatic cancer therapy. MITO@HA binds to sodium hyaluronate, which is highly expressed in pancreatic cancers. This study seeks to evaluate MITO@HA treatment efficacy, clarify its inhibitory effect on pancreatic cancer, and provide an experimental basis for the use of organic polymer nanoparticles loaded with antitumor drugs to treat pancreatic cancer. This treatment strategy was developed for pancreatic cancer based on the hydrophobic behavior of the nanopharmaceutical MITO@HA. The average particle size of MITO@HA was 51.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 nm, and the particles had a spherical structure. CCK-8 assays revealed that both MITO and MITO@HA inhibited the proliferation of pancreatic cancer cells. that the most suitable experimental conditions were determined to be exposing pancreatic cancer cells to 0.5 uM/L MITO@HA for 2 days. PANC-1 pancreatic cancer cells and pancreatic cancer tissues were found to express high levels of CD44. In in vitro experiments, MITO@HA inhibited G0/G1 phase arrest, increased apoptosis, and decreased cell replication, cell migration and invasion in the pancreatic cancer cell cycle compared to MITO alone. Therefore, we believe that MITO@HA has a good tumor cell inhibitory effect. Furthermore, in vivo experiments revealed that the tumor volume in nude mice in the MITO@HA group decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and both MITO and MITO@HA treatment decreased the tumor growth curves, with MITO@HA decreasing them more than MITO alone. Compared with those in the control group and the MITO group, the HE staining of tumors in the MITO@HA group showed massive liquefaction necrosis of the tumor tissues. Safety evaluation of the nude mice in the MITO@HA group revealed that the mice had a normal blood profile, normal liver and kidney function, and normal myocardial enzymes. The above results indicate that MITO@HA can effectively accumulate in pancreatic cancer tumor tissue through the EPR effect and CD44 receptor targeting, leading to liquefaction and necrosis of tumor tissue, thereby effectively reducing tumor growth. The above results showed that MITO@HA is highly safe and can enhance the antitumor effect on pancreatic cancer, providing an experimental basis for clinical application.\u003c/p\u003e","manuscriptTitle":"Drug-loaded MITO@HA nanodrugs for evaluating the efficacy of targeted therapy for pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-26 05:56:34","doi":"10.21203/rs.3.rs-3972887/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"949ca966-4669-4006-8d98-a62b48c7eb38","owner":[],"postedDate":"February 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-15T08:54:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-26 05:56:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3972887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972887","identity":"rs-3972887","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}