Synthesis, characterization, and application of self-assembled safflower polysaccharide nanoparticles as liver targeting drug delivery carrier

Synthesis, characterization, and application of self-assembled safflower polysaccharide nanoparticles as liver targeting drug delivery carrier | 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 Article Synthesis, characterization, and application of self-assembled safflower polysaccharide nanoparticles as liver targeting drug delivery carrier Haotian Bai, Jing Yang, Junhao Zhang, Rui Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6119667/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 Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death with chemotherapy and traditional surgery showing limited effectiveness. The present work aimed to study the feasibility of safflower polysaccharide (SPS) as an instinctive liver-targeting drug delivery carrier with applications in HCC. SPS-polyethyleneimine (SPS-PEI), hyaluronic acid-SPS-polyethyleneimine (HA-SPS-PEI), and hyaluronic acid-folic acid-SPS-polyethyleneimine (HA-FA-SPS-PEI) conjugates were synthesized by an esterification reaction and characterized by conventional methods. SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (SPNPs, HSPNPs, and HFSPNPs, respectively) and siRNA-loaded SPNPs, HSPNPs, and HASPNPs (siRNA FAM /SPNPs, siRNA FAM /HSPNPs and siRNA/HASPNPs, respectively) were fabricated with a roughly spherical shape, with sizes were ranging 100 ~ 200 nm in aqueous solution. Compared with free siRNA FAM , siRNA FAM /HASPNPs displayed enhanced serum stability, hypo toxicity, and a sustained release of siRNA FAM over 64 h. In the in vivo cellular uptake behavior study, the HASPNPs showed excellent HCC tumor-targeting capability because of the specific recognition by the folic acid and hyaluronan receptors (CD44) overexpressed on the HCC tumor membrane. The tissue staining of siRNA FAM /HASPNPs in mice further demonstrated that HASPNPs could distinctly enhance the distribution of siRNA FAM into the HCC tumor. Our results indicate that HASPNPs may serve as a promising HCC tumor-targeting drug delivery carrier for HCC prevention. Biological sciences/Cancer Biological sciences/Drug discovery Dual-target Safflower polysaccharide Hepatocellular carcinoma Self assembled nanoparticles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction Hepatocellular carcinoma (HCC), one of the most widespread malignancies with the highest prevalence, is the second most common cause of cancer-related death [ 1 ]. Chemotherapy and traditional surgery have serious side effects and do not produce the best outcomes. In recent years, RNA interference (RNAi) technology has become a popular gene interference technique and an emerging cancer treatment approach in addition to surgery, chemotherapy, and radiation therapy. Strong selectivity, good specificity, quick action, and high gene silencing efficiency are all advantages of using RNAi technology for the treatment of hepatocellular carcinoma. This method may be used to decrease the expression of some crucial oncogenes and key signaling pathway molecules in tumor cells, thereby preventing the growth of tumors [ 2 ]. Researchers' interest in natural polysaccharides, a secure, effective, and low-toxic gene carrier, has gradually grown. After proteins and nucleic acid, polysaccharides are the third type of macromolecule that stores biological information. Natural polysaccharides continue to garner interest in the realm of biological materials due to their high non-toxicity, good biocompatibility, and biodegradability [ 3 ]. Safflower polysaccharide (SPS) is a naturally occurring polysaccharide obtained from Compositae Carthamus tinctorius L. It has anti-tumor, antioxidant, and immunity-boosting characteristics and also contains the active group -OH. The synthesis of SPS with various biological effects can therefore be made possible by the chemical alteration of -OH. Polyethyleneimine (PEI) is a classical transfection agent with a high transfection rate, exhibiting good biocompatibility and low toxicity. Owing to their joint advantages, researchers used the two materials to modify the natural polysaccharide with PEI as the gene carrier [ 4 ]. Cell internalization may be accelerated by identifying polysaccharide receptors on the cell surface. Cationic polysaccharide carriers have advantages over other cationic polymers concerning low cytotoxicity and immunogenicity, good biodegradability, and high water solubility [ 5 ]. The receptor-mediated targeted gene delivery system is the most advanced cell-specific vector system with the advantages of high specificity and high affinity, and it greatly improve the drug delivery efficiency of drugs [ 6 – 7 ]. The commonly used specific receptors include hyaluronic acid (HA), folic acid (FA), integrin, and galactose receptors. As a new targeted drug delivery strategy, dual-receptor-mediated targeted drugs could deliver nanoparticles to specific sites more effectively. Therefore, the dual-receptor rake strategy has been widely used in drug delivery systems in recent years [ 8 ]. Bone morphogenetic proteins (BMPs) are a group of functional proteins that are widely involved in biological processes that regulate the proliferation, differentiation, and apoptosis of various cells. BMP expression is involved in the formation of embryos and organs, as well as in the formation and development of tumor diseases. BMP7 is a member of the transforming growth factor beta (TGF-β) superfamily. An increasing number of studies have shown that BMP7 is significantly expressed in various epithelial tumors, such as breast cancer, primary melanoma, prostate cancer, and colorectal cancer [ 9 – 11 ]. However, the relationship between BMP7 gene changes and malignant biological characteristics of hepatocellular carcinomas, such as malignant differentiation degree, proliferation, invasion, metastasis, and recurrence, is rarely reported in local and international research. Based on the above studies, cationic safflower polysaccharide (SPS-PEI) was selected as the basic gene carrier in this study, and siRNA was bound by electrostatic adsorption. HA and FA were covalently combined to form a polymer through esterification reaction, which was then coated with SPS-PEI to improve delivery efficiency. While shielding the positive charge to reduce cytotoxicity, FA and HA receptors (CD44) on SMMC-7721 cell surfaces could be simultaneously targeted to construct a double-targeted nano gene vector. 2. Materials and methods 2.1. Materials The plant material collected was identified as the dried tubular flower of the Compositae C. tinctorius L. by Professor Rui Wang (College of Pharmacy, Heilongjiang University of Chinese Medicine) at the Medical Botany Center of Heilongjiang University of Chinese Medicine, where a voucher specimen (No. 20210493) has been deposited. Polyethyleneimine (PEI), N, N-carbonyl imidazole (CDI), tetramethylsilane (TMS), folic acid (FA), hyaluronic acid (HA), N-butyl hydroxy (NHS), dicyclohexyl-carbodiimide (DCC), and anti-fluorescence quenching (AFQ) were obtained from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Annexin V-FITC/PI double staining kit, AO Staining, Hoechst33258 and protein marker were obtained from Sangon Biology Co. Ltd. (Shanghai, China). BMP-7, β-actin, and Goat anti-rabbit Immunoglobulin G (IgG) (H&L) were purchased from Abways Company (Shanghai, China). Detailed information about siRNA can be found in Table 1 . SiRNA FAM was synthesized by Suzhou Jima Gene Co. Ltd. (Suzhou, China). Table 1 Sequence of siRNA Name Upstream primers Downstream primers siBMP-7 CCG UCC UCU ACU UCG AUG AdTdT UCA UCG AAG UAG AGG ACG GdTdT siRNA FAM UUC UCC GAA CGU GUC ACG UdTdT ACG UGA CAC GUU CGG AGA AdTdT β-actin CTC CAT CCT GGC CTC GCT GT GCT GTC ACC TTC ACC GTT C 2.2. Cell lines and cell culture The human hepatocellular carcinoma cell (SMMC-7721) and the normal human hepatic cell (LO2) were obtained from Shanghai Fuheng Biotechnology Co. Ltd. (Shanghai, China). SMMC-7721 cells and LO2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Vivacell, Shanghai, China) supplemented with 10% (v/v) fetal bovine serum (Hyclone) and antibiotics (100 U/ mL penicillin and 100 U/mL streptomycin) (Hyclone) at 37°C in a humidified atmosphere containing 5% CO 2 . 2.3. Animals Female C57BL/6 mice that were specifically pathogen-free (SPF) and weighed (20 ± 2) g were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Shenyang, China).The mice were housed in polypropylene cages at 25 ± 1°C at a relative humidity of 50 ± 20% and a 12-h light-dark cycle with free access to food and water. The study was approved by Ethics Committee of Heilongjiang University of Chinese Medicine (approval number:202208011). All methods were carried out in accordance with relevant guidelines and regulations. This study was carried out in compliance with the ARRIVE guidelines. 2.4. Separation and purification of safflower polysaccharides The unrefined SPS was extracted according to a method used in a previous study [ 12 ]. The specific operational details are presented in Supplement 1. The phenol-sulfuric acid method was used for color tracking, and the absorbance value was measured at 490 nm. Polysaccharide samples with the same elution peak were collected. The elution curve was plotted using the tube number as abscissa ( X ) and absorbance (A) as ordinate ( Y ). Components were separated and purified form Sephadex G-100 and prepared into a 1 mg/mL solution after dialysis, concentration, and freeze drying. Afterwards, the solution was scanned using the UV spectrophotometer within the wavelength range of 200～800 nm. Distilled water was used as the blank control. The characteristic absorption peaks of nucleic acid (260 nm) and protein (280 nm) were detected. The separation and purification flow chart is shown in Fig. 1 . 2.5. Structural identification of safflower polysaccharide 2.5.1. Determination of total carbohydrate and uronic acids Glucose and glucuronic acid control solutions with concentrations of 20, 40, 60, 80, and 100 µg/mL were prepared. Standard curves were drawn using glucose concentration as abscissa ( X ) and absorbance as ordinate ( Y ). SPS-1 and SPS-2 were accurately weighed to prepare their respective 100 µg/mL aqueous solutions. Total polysaccharides and uronic acids were determined using sulfuric acid-anthracone and sulfuric acid-carbazole method. 2.5.2. Determination of relative molecular mass The relative molecular weights (4600～133800) of the dexglucoside series of reference substances were added to distilled water to prepare a 1 mg/mL solution, and then filtered using a 0.45 µm microporous membrane. Then 500 µL of each of the aforementioned reference solution was taken in turns, starting from small to large molecules. Three replicates of each sample were taken in parallel, and the retention time of their elution peaks were recorded under the known chromatographic conditions. The standard curve was derived from the logarithm of the molecular weight (M W ) of the reference substance and its corresponding retention time (tR), and the linear regression equation was lgM W = a + btR. SPS-1 and SPS-2 prepared using the aforementioned method were treated with 50 µL and determined according to the same method, and the retention time (tR) of the polysaccharide was recorded. The weight average molecular weight, Mw, number average molecular weight, Mn, and polydispersion index, D, of polysaccharides were calculated according to the known standard curve. 2.5.3. Analysis of monosaccharide composition The monosaccharide components were analyzed using pre-column derivatization high performance liquid chromatography (HPLC). The purified 50 mg SPS-1 and SPS-2 were respectively weighed in a hydrolysis tube, and 1 mL water and 1 mL 4 mol/L trifluoroacetic acid were added. Under conditions of nitrogen protection, the water was hydrolyzed at 110°C for 2 h and then cooled to room temperature. Then 0.1 mL was placed in a 4 mL centrifuge tube, and vacuum dried at 60°C for 2 h. The standard monosaccharides were obtained by sequentially adding 5 mg of D-glucose, D-galactose, D-galacturonic acid, D-xylose, D-mannose, L-rhamnose, and L-arabinose to a 25 mL volumetric flask. In addition, 500 µL of the standard monosaccharide solution and 2 hydrolyzed polysaccharide samples were placed in a centrifuge tube. Then 0.05 mL 0.3 mol/L NaOH and 0.05 ml PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution were added to each tube. The reaction was performed in a water bath at 70°C for 60 min under nitrogen protection and cooled to room temperature. Then 0.5 mL 0.3 mol/L HCl, 0.75 mL H 2 O, and 1.5 mL chloroform were added for extraction. The chloroform layer was discarded and extracted three times. The water layer was combined with PMP-derived products on the upper layer and filtered by means of a 0.45 µm filter membrane. The samples were directly injected for gas chromatography (GC) analysis. 2.5.4. FT-IR analysis In order to perform the FT-IR study, 3 mg of SPS-1 and SPS-2 were ground and compressed using KBr. A total of 24 scans were performed within the 4000～400 cm − 1 range. 2.5.5. 1 H-NMR analysis Prior to NMR analysis, 30 mg of over-dried polysaccharide was dissolved in 0.5 ml of 99.97% D 2 O for 6 hours after being repeatedly freeze-dried in D 2 O to exchange deuterium. On a Bruker AV-500 spectrometer (Bruker, Rheinstetten, Germany), the 1H- was acquired at 30°C at 500 MHz for 1 H. The acquisition time (AT = 0.5 s), sweep width (SWH = 20,000 Hz), relaxation delay (d1 = 10 s), and 90° pulse time (p1 = 2.5 µs) were the parameters that were specified for the 1 H-NMR spectra. The recordings were made with a relaxation delay of 1.5 s. Chemical shifts are measured in parts per million (ppm) and are calibrated internally using acetone, which has a δ31.21 ppm for carbon and a δ2.19 ppm for hydrogen. The TopSpinTM3.5 program from Bruker was used to evaluate the data. 2.5.6. Structure analysis of Congo red Solutions of Congo red (0.2 mmol/L) and SPS-1/2 (2 mg/mL) were made. After thoroughly mixing 1.5 mL of Congo red solution with 1 mL of polysaccharide solution and 0.5 mL of water, the mixture was split into 10 equal portions. Concurrently, the aqueous solution containing polysaccharides was converted to an aqueous solution and designated as the Congo red blank control group. For around ten minutes, the solution was incubated at room temperature. At a wavelength of 200 ~ 600 nm, UV scanning was carried out, and the highest absorption wavelength was noted. 2.5.7. Periodic acid oxidation experiment First, a consistent volume of 15 mmol/L potassium periodate (KIO 4 ) original solution was created, and its concentration was diluted to 10, 15, 30, 45, and 60 µmol/L KIO 4 solution. The UV absorbance was then measured at 223 nm. KIO 4 concentration was represented by the x-coordinate ( X ) on the standard curve, and absorbance value by the y-coordinate ( Y ). To stop the light reaction at room temperature, an additional 5 mg of SPS was weighed and 10 mL of KIO 4 solution (15 mmol/L) was added. Subsequently, a volume of 25 mL was maintained while 100 µL was taken at 0, 4, 8, 12, 24, and 48 h. The absorbance value at 223 nm was obtained using distilled water as the blank control. The absorbance value steadily dropped as KIO 4 was used up until the measured value steadied, signifying that the reaction was finished and allowing for the calculation of KIO 4 consumption. In order to use up the extra KIO 4 , 1 mL of glycol was added last, and the reaction was stopped by shaking for 10 minutes. To ascertain the amount of formic acid produced, 2 mL of the previously described oxidized solution was added along with 2 drops of phenolphthalein indicator, and the mixture was titrated with 0.01 mol/L NaOH. 2.6. Synthesis and characterizations of the SPS-PEI conjugate As per an earlier study, the SPS was isolated from the dry safflower flowers and refined. An esterification reaction could bond PEI to SPS [ 13 ]. The specific operational details are presented in Supplement 2. Using DMSO d6 as the solvent at 400 MHz, FT-IR spectroscopy Nicolet 170SX and 1 H-NMR spectrometry were used to determine the chemical structures of SPS and SPS-PEI. By employing fluorescence spectroscopy with pyrene as a hydrophobic probe, the critical aggregation concentration (CAC) of the SPS-PEI conjugate was used to measure its self-aggregation characteristic. In summary, a succession of volumetric flasks were filled with a known quantity of pyrene in acetone, and the acetone was blown out using N 2 . Following the formation of a pyrene film, different concentrations of the SPS-PEI conjugate solution were made and put into each volumetric flask, yielding a final solution with a pyrene concentration of 6 × 10 − 7 M. To acclimate the pyrene and conjugate, the mixture was shaken for 12 h at 37°C in a thermostatic shaking water bath. With an emission wavelength of 372 nm, the fluorescence excitation spectra were obtained using a F7000 fluorescence spectrometer operating in the 300 ~ 360 nm range. For the purpose of measuring the intensity ratio, the fluorescence intensities (I 339 and I 335 ) at 339 and 335 nm were noted. 2.7. Synthesis and characterizations of the siRNA/SPNPs, siRNA/HSPNPs, and siRNA/HFSPNPs An esterification reaction could bind HA to FA. In short, 1.0 g DCC and 288 mg NHS were dropped and activated at 30°C for 5 h after 50 mg FA was dissolved in 3 mL DMSO at room temperature for 30 min. The activation process produced precipitation, which was filtered out. Moreover, 10 mL of formamide containing 100 mg of HA was heated to 40°C to fully dissolve it, and the mixture was added drop by drop over the course of 48 h [ 14 ]. Filtration was used to remove the precipitate, and for 48 h, light protection was used while dialyzing with deionized water. The HA-FA conjugate was obtained by lyophilizing the water, which was replaced every 6 h. The FT-IR and 1 H-NMR analysis procedure used for HA-FA was the same as that used for SPS. HA-SPS-PEI and HA-FA-SPS-PEI were produced using the magnetic-stirring technique depicted in Fig. 2 . In short, SPS-PEI was gradually added to the aqueous solutions of HA and HA-FA that were magnetically agitated and set at 1 mg/mL for a duration of 15 min. Thus, HA-SPS-PEI and HA-FA-SPS-PEI were acquired. The dialysis-sonication approach was used to prepare SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (SPNPs, HSPNPs, and HFSPNPs). In summary, 10 mg of SPS-PEI, HA-SPS-PEI, or HA-FA-SPS-PEI was dissolved in 1 mL of DMSO and then gradually added to 3 mL of deionized water while being continuously stirred at room temperature (1000 rpm). The combination was dialyzed against deionized water for a day. A well-dispersed nanoparticle solution was then achieved by sonicating the mixture for 3 min. Zeta potentials of complexes produced at a mass concentration of 1 mg/mL and dissolved in an appropriate PBS solution were measured using a Nano90 Malvin Laser at 25°C and a 90° scattering angle using dynamic light scattering (DLS). With the exception of adding various quantities of siRNA FAM to the SPS-PEI DMSO solution, the same protocol was used to generate the siRNA FAM -loaded SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (siRNA/SPNPs, siRNA/HSPNPs, and siRNA/HFSPNPs). Centrifugation was used to extract free siRNA for 15 min at 3000 rpm. After lyophilization, siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs were finally produced. In order to assess the loading of siRNA in SPNPs, HSPNPs, and HFSPNPs, a predetermined volume of lyophilized siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM / HFSPNPs was dissolved in an appropriate PBS solution. The fluorescence intensity value was then measured by adding Ribogreen fluorochrome (x200) and TBE solution. With the assistance of a spectrofluorometer set to measure at 436 nm, the siRNA FAM encapsulation efficiencies (EE) of SPNPs, HSPNPs, and HFSPNPs were computed as follows: EE (%)= \(\:\frac{\text{C}\text{t}\text{o}\text{t}\text{a}\text{l}-\text{C}free}{Ctotal}\) The siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs in PBS solution (pH 7.4) at 1 mg/mL were subjected to DLS analysis at 25°C using a Nano 90 Malvern Laser to determine their mean particle size, polydispersity index (PDI), and zeta potential. In order to investigate the morphology of the nanoparticles in more detail, the sample solutions (1 mg/mL) were placed onto copper grids covered with carbon, negatively stained for 2.5 min with a solution of 1% phosphotungstic acid, and allowed to air dry at room temperature. An H-7000FA Transmission Electron Microscope was used to image the nanoparticles' ultrastructure. 2.8. Biocompatibility investigation of blank nanoparticles Indicators for assessing the biocompatibility of the SPNPs, HSPNPs, and HFSPNPs included serum stability, cytotoxicity, and buffer capacity. For the serum stability test, 100 µL of FBS was combined with 0.9% NaCl (negative group) or the same volume of SPNPs, HSPNPs, and HFSPNPs solution at different concentrations. A UV-Vis spectrophotometer was used to measure the absorbance of each group at 630 nm at 0, 2, 6, 12, 18, and 24 h after incubation in a water bath at 37°C away from light. By computing the absorbance ratio between the samples and the negative group at each time point, the relative turbidity was determined. After being seeded at a density of 3 × 10 3 cells/well in 96-well plates, LO2 and SMMC-7721 cells were cultivated for a whole night. Upon being treated for 96 hours with 0, 1, 10, 20, 40, 80, or 160 µg/mL of SPNPs, HSPNPs, and HFSPNPs, 20 µL of MTT (5 mg/mL) was added to each well, and the mixture was incubated for an extra 4 h at 37°C. Following the formation of formazan crystals, 150 µL of DMSO was added, and a microplate reader was used to measure the absorbance at 570 nm. Wells devoid of any cells served as the blank, and the untreated cells served as the control. Five analyses were conducted on each sample at each concentration. The following formula was used to determine cell viability (%) = \(\:\frac{\text{A}570（treated）-\text{A}0}{\text{A}570（untreated）-\text{A}0}\) where A 570(treated) is the absorbance of cells treated with samples, A 570(untreated) is the absorbance value of the untreated cells, and A 0 is the absorbance value of the blank medium. The buffer capacity of the SPNPs, HSPNPs, and HFSPNPs was investigated via acid-base titration. Following the dispersion of SPNPs, HSPNPs, or HFSPNPs (0.2 mg/mL) in the aqueous NaCl solution (0.15 mol/mL), an aqueous NaOH solution (0.1 mol/mL) was added until the pH reached 10. Then, a solution of 0.1 mol/mL HCl (3–5 µL) was added. The pH of the mixture was determined using a microprocessor pH meter following each addition of the HCl solution. The 0.2 mg/mL deionized water was regarded as the positive control and the 0.15 mol/mL NaCl solution as the negative control. 2.9. In vitro drug release study Utilizing a dynamic dialysis procedure in PBS (pH 7.4) containing 0.5% Tween 80 and 10% ethanol, the siRNA FAM release capacity from SPNPs, HSPNPs, and HFSPNPs was investigated [ 15 ]. In a nutshell, 50 mL of the release media were dialyzed against 1 mL of free siRNA and siRNA FAM /SPNPs, siRNA FAM /HSPNPs, or siRNA FAM /HFSPNPs solution at 37°C in a shaking incubator at 120 rpm. The release medium was swapped out on a regular basis for equivalent amounts of brand-new release medium. Every experiment was conducted in triplicate, and the amount of released siRNA was determined using a fluorescence spectrometer. 2.10. Study on the pharmacodynamics characteristics of SPNPs, HSPNPs, and HFSPNPs 2.10.1. In vitro cellular uptake characteristics of nanoparticles In order to confirm that the nanoparticles could target the liver, SPNPs, HSPNPs, and HFSPNPs were labeled with siRNA FAM , a simulated fluorescent marker. LO2 cells and SMMC-7721 cells were seeded on coverslips in 24-well plates for the qualitative cellular uptake study. The cells were then separated into the following groups: free siRNA FAM , siRNA FAM /SPNPs, siRNA FAM /HSPNPs, siRNA FAM /HFSPNPs, and siRNA FAM /HFSPNPs with the antagonist HA (or FA) (HA and FA pretreatment at a concentration of 1 mg/mL). The cells were exposed to new medium containing free siRNA FAM or siRNA FAM nanoparticles (siRNA FAM concentration, 100 nM) for 1, 2, or 4 h following 24 h of cell attachment. After discarding the media, the cells underwent three rounds of cold PBS washing, were fixed for 10 min using cold 4% paraformaldehyde, and their nuclei were then stained for 20 min with 4′,6-diamidino-2-phenylindole (DAPI) in order to identify them. The coverslips were then gently taken out of the wells and put onto the glass slides. A confocal laser scanning microscope was used to take the pictures. Six-well plates were used to seed LO2 cells for the quantitative cellular uptake investigation. Following a 24-hour incubation period, the cells were subjected to several siRNA FAM forms utilizing the identical procedure as the CLSM investigation. As a negative control group, cells that were not given medication were employed. Three cycles of cold PBS washing followed by trypsinization and 3 min at 1300 rpm centrifugation were performed on the cells. The cells were centrifuged after being cleaned with PBS. The cells were subsequently identified using flow cytometry after being resuspended in 500 µL of PBS solution. 2.10.2. In vitro gene silencing SMMC-7721 cells were cultivated in 2 mL of DMEM with 10% FBS for 24 h after being plated at a density of 4×10 4 cells per well in 6-well plates. The cells were allowed to incubate for 4 h before being incubated for a further 20 h with fresh medium after the medium was replaced with opti-MEM containing siRNA FAM /HFSPNPs (1:1, 5:1, 10:1, and 20:1). The lysis buffer was used to extract the transfected cell proteins. Proteins were separated via gel electrophoresis after being put in equal amounts into the wells of a 10% SDS-PAGE gel. Upon transferring the proteins from the gel to the polyvinylidene difluoride membrane, the membranes were blocked for 60 min with 5% BSA. The membranes underwent 16 h of incubation at 4°C with BMP7 rabbit antibody (1:1000) and β-actin rabbit antibody (1:5000), followed by 60 min of secondary antibody treatment. Applying an enhanced ECL detection technique, the expression of BMP7 was identified. A Mini Chemi610 Imaging System was used to identify the western blot signals. Fig. S1 displayed the original western blots. 2.10.3. Protective studies of siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs in HCC tumor After being collected, SMMC-7721 cells were suspended in PBS at a density of 1×10 7 cells/mL. In order to initiate an in vivo focused experimental inquiry, female C57BL/6 mice were infected with 0.1 mL cell solution in the axilla until the tumor volume expanded to (100 ± 20) mm 3 . 4 groups (n = 5) of mice were randomly assigned: 1. control group (saline); 2. siBMP7/SPNPs; 3. siBMP7/HSPNPs; and 4. siBMP7/HASPNPs. Every 2 days, an intravenous injection of 200 µL of either the standard saline or the various siBMP7 formulations (equal to 0.33 mg/kg/day) was given into the tail four times in a row. Following the fourth dosage, the mice were put to sleep after an 8-hour fast (Fig. 3 ). The mice's primary organs and tumor tissues were completely removed, and the tumor tissues were weighed. For further H&E staining, all tissues were fixed in a 10% formalin solution. To identify the apoptosis of SMMC-7721 cells, cryostat sections of the tumor were cut into 10 µm thick sections and stained with Acridine orange (AO). These sections were then seen using a Leica DM2500 fluorescence microscope. 2.10.4. Western blotting analysis For western-blot analysis, RIPA buffer with 1 mM PMSF and 1% protease and phosphatase inhibitors were used to lyse the frozen tumor samples. The total protein concentration was assessed by the BCA method, and equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Incubation with the primary antibodies against BMP7 (1:1000) and β-actin (1:4000) was employed at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. Densitometry using ImageJ software was calculated for quantification, and the densitometry results were normalized relative to the β-actin bands. The original western blots were shown in Fig. S1 . 2.10.5. In vitro safety evaluation For in vitro safety evaluation, the tumor tissues and major organs of mice were dissected and fixed in 4% paraformaldehyde at room temperature for 24 h. Sections were prepared by paraffin embedding and H&E staining and observed and photographed under a CKX41 Optical microscope. 2.11. Statistical analysis All experimental data were performed at least in triplicate and expressed as mean ± standard deviation. Statistical comparisons between groups were carried out using a one-way analysis of variance followed by the Student’s t-test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Separation, purification and chemical compositions of SPS After 95% ethanol extraction, 5% alkali extraction, ethanol precipitation, and deproteinization, 37.7 g of crude polysaccharide SPS (yield 7.54% of 500 g of dry material) was produced from dried safflower. Following SPS fractionation using DEAE-cellulose ion exchange chromatography (Fig. 4 A), two glycoconjugates, known as SPS-a and SPS-b, were obtained and eluted at distilled water and 0.25 M NaCl, respectively. A SephadexG-100 column was used to further purify SPS-a and SPS-b in order to produce a significant polysaccharide fraction known as SPS-1 (2.92 g, yielding 0.58% of 500 g dried material) and SPS-2 (1.72 g, yielding 0.34% of 500 g dried material)(Fig. 4 B and Fig. 4 C). For the next physicochemical and animal tests, the peaks of SPS-1 and SPS-2 were collected, dialyzed, and dried. Purified polysaccharide SPS-1 and SPS-2 have respective carbohydrate contents of 94.2% and 92.1%. The absence of an absorption peak in the UV spectra of SPS-1 between 200 and 280 nm suggests that the polysaccharide lacked protein and nucleic acid (Fig. 5 B). In the present study, we focused on the SPS-1 (subsequently replaced by SPS) fraction due to its easy access to enrich. Standard curves of glucose and uronic acid are all shown in Fig. 5 A. The composition of the monosaccharide can be determined by the retention time. As shown in Fig. 5 C、 Tab. and Table 4 , the derivatives of monosaccharide standard D-glucose (Glu), D-galactose (Gal), D-xylose (Xyl), D-galacuronic acid (Gala), D-mannose (Man), L-rhamnose (Rha) and L-arabinose (Ara) showed absorption peaks at about 26.025, 29.398, 30.700, 22.450, 12.759, 17.051 and 32.137min, respectively. By comparing SPS-1 and SPS-2 polysaccharide maps, the monosaccharide composition can be determined by corresponding to them near the partially identical position, and the molar ratio of each monosaccharide can be calculated from the retained peak area. SPS-1 monosaccharide components respectively Man, Glu, Xyl, Ara, Gal, Rha, the mole ratio is: 5.36:8.72:4.61:1.96:19.36:4.52. The monosaccharide components of SPS-2 were Rha, Glu, Gal, Ara and Man with molar ratios of 3.00:7.12:5.88:1.16:4.02. The molecular weight of polysaccharide chromatogram is shown in Fig. 5 D(a)&(b) and Table 2 . The retention time of SPS-1 was 20.269 min, the retention time of SPS-2 was 17.789 min, the MW of SPS-1 was 5546Da, Mn was 2984Da, and the polydispersis (D) was 1.925. Mw of SPS-2 is 30902Da, Mn is 13700Da, and D is 2.440. The results show that the larger the polydispersity, the wider the molecular weight distribution. This value is less than 1.05, where the sample is monodisperse, and greater than 1.05, where the sample is polydisperse. Therefore, both SPS-1 and SPS-2 are polydisperse samples, that is, the sample is widely distributed. Studies had shown that if there is a three-dimensional helical structure in the polysaccharide molecule, it will form a complex with Congo red solution, resulting in the shift of the maximum absorption wavelength of Congo red solution. As shown in Fig. 5 E, the maximum absorption wavelength of SPS-1 is close to that of Congo red phase, so there is no 3D helix structure, while the maximum absorption wavelength of SPS-2 shows a red shift, which is higher than that of Congo red, indicating that a complex can be formed. Thus, SPS-2 has a 3D helix structure. The results of infrared measurement of SPS-1 are shown in Fig. 5 F. There is a strong absorption peak at 3423cm − 1 , indicating that there are intermolecular and internal hydrogen bonds of SPS-1. The absorption peak at 2924cm − 1 is the special absorption peak of saccharide substances, 1425cm − 1 is caused by C-H angular vibration, 1051cm − 1 and 950cm − 1 are C-O-C absorption peaks, 921.97cm − 1 is the stretching vibration of the ring, and 895.45cm − 1 is the C-H angular vibration of pyranose. It was judged to be the residue absorption peak of β-D-man. The infrared measurement results of SPS-2 are shown in Fig. 5 F, 3423cm − 1 has a smooth absorption peak, SPS-2 has intermolecular and internal hydrogen bonds, 2920cm − 1 is the peak of C-H tensile vibration, is the absorption peak of saccharide, 1660cm- 1 is the absorption peak of C = O in -COOH. 1433cm − 1 is the C-O stretching vibration in -COOH, 1050cm − 1 has an absorption peak, indicating that SPS-2 has pyanose, 900 ~ 1225cm − 1 has an absorption peak of C-O-C and -OH, 917cm − 1 is the characteristic absorption peak of α-D-gal. 841cm − 1 is a C-H stretching vibration, which indicates that SPS-2 has α-D-galactopyranose. As shown in Fig. 5 G, the absorption signal peak of SPS-1 near δ4.79 ppm is the tritium proton signal in solvent D 2 O, and the proton peak signal at δH4.69 ppm indicates that it belongs to the sub-peak signal of β-D-glu residue matrix. The concentration area of the signal peak is less than 5.0 ppm, indicating that it is a β-configuration glycosidic bond, but there is an inverted peak. This is in agreement with the IR spectroscopic results. The δH1.18 ppm and 1.16 ppm resonance signals in the high-field region were inferred to be a small amount of rhamnose signal peak. SPS-2 is similar to SPS-1. The absorption signal peak near 4.79 ppm is the tritium proton signal in solvent D 2 O, and δH5.06 ppm belongs to the proton signal of Man, indicating that SPS-2 belongs to the alpha configuration glycosidic bond, which is consistent with the infrared spectrum results. The δH1.23 ppm and δH1.16 ppm in the high-field region may be the resonance methyl proton signal peak, which is a trace of rhamnose signal. Standard curve of KIO 4 is shown in Fig. 5 H.SPS-1 and SPS-2 can be completely reacted by KIO 4 within 2 days, and the consumption of KIO 4 and the production of formic acid are shown in Table 5 . The amount of CH 2 O 2 produced by SPS-1 was 13.2 µmol, indicating that for every 1→6 bonded glycosyl, the amount of 1→2 or 1→4 bonded glycosyl was 27.3 µmol, and the remaining 1→3 bonded glycosyl was 9.5 µmol. In SPS-1, the 1→6 linkage or non-reducing terminal group accounted for 26.4%, other oxidable 1→2 or 1→4 linkage glycosyl accounted for 54.6%, and the non-oxidable 1→3 residue accounted for 19%. These results suggested that SPS-1 was mainly composed of glucans with 1→2 or 1→4 linkage glycosyl as the main chain. The amount of CH 2 O 2 produced by SPS-2 was 26.12 µmol, indicating that for every 1→6 bonded glycosyl, the amount of 1→2 or 1→4 bonded sugar group was 4.11µmol, and the remaining 1→3 bonded glycosyl was 19.77 µmol. In SPS-2, the 1→6 glycoside-linked or non-reducing terminal group accounted for 52.24%, the 1→2 or 1→4 glycoside-linked group accounted for 8.22%, and the non-oxidable 1→3 residue accounted for 39.54%, indicating that SPS-2 was mainly composed of 1→6 glucans bonded to the main chain. Secondly, ara-gal with the glycosyl main chain partially bonded at the 1→3 position is present. The specific connection mode of SPS-1 and SPS-2 needs to be determined after Smith degradation and methylation. Table 2 Molecular weight and polydispersion index of SPS-1 and SPS-2 Sample Mw(Da) Mn(Da) D SPS-1 5546 2984 1.925 SPS-2 30902 13700 2.440 Table 3 Analysis of SPS-1 monosaccharide composition Name Retention time Response factor Peak area Content(mg/L) Man 12.832 155.140 10549.999 163.362 Rha 17.168 119.512 9808.048 137.762 Glu 26.124 169.428 21132.109 265.768 Gal 29.786 165.248 40087.860 590.054 Xyl 31.030 173.850 8488.045 140.503 Ara 32.424 182.093 5880.004 48.76 Table 4 Analysis of SPS-2 monosaccharide composition Name Retention time Response factor Peak area Content(mg/L) Man 12.805 155.140 15285.930 168.530 Rha 17.114 119.512 11761.550 128.413 Glu 26.083 169.428 18316.426 209.085 Gal 29.585 165.248 16267.695 198.444 Ara 32.284 182.093 7859.102 48.077 Table 5 Oxidation of KIO 4 Sample Added amount Consumption of KIO 4 Production of CH 2 O 2 1→6 amount of glycosyl bonded 1→2/1→4 amount of glycosyl bonded 1→3 amount of glycosyl bonded SPS-1 50 53.7 13.2 13.2 27.3 9.5 SPS-2 50 56.35 26.12 26.12 4.11 19.77 3.2. Synthesis and characterizations of the SPS-PEI conjugate The hydrophobic moiety CDI was selected to modify the water-soluble SPS and produce the amphiphilic SPS-PEI compound. To sum up it briefly, PEI was first created to functionally introduce the carboxylic acid (-COOH), and as shown in Fig. 6 A, it was subsequently conjugated with SPS through an esterification reaction. The 1 H-NMR and FT-IR analyses verified the chemical structure. The 1 H-NMR spectra for SPS-PEI, as displayed in Fig. 6 C, revealed additional peaks at 2.52 ~ 2.74 ppm that were attributed to the protons of PEI in addition to the distinctive SPS peaks between 3.7 and 5.3 ppm, indicating the successful attachment of PEI to SPS. FT-IR was used to confirm the synthesis of SPS-PEI in more detail. The FT-IR spectra of SPS and SPS-PEI, as shown in Fig. 6 B, both showed a distinctive absorption band about 3400 cm − 1 , which is due to the stretching vibrations of the hydroxyl group (-OH). Nevertheless, the intensity of this band is much lower than that of SPS, suggesting that some hydroxyl groups react. When comparing SPS with SPS-PEI, the former revealed a characteristic deformation peak at roughly 1570 cm − 1 , while the latter revealed a new peak at about 1700 cm − 1 , linked to the stretching vibrations of the newly created ester carbonyl group (C = O). Consequently, the synthesis of the SPS-PEI conjugate was accomplished. When the conjugate begins to self-assemble into nanoparticles by intra/intermolecular interaction, the threshold concentration can be used to estimate the critical aggregation concentration (CAC). Pyrene was used as the probe in the fluorescence probe technique, which was used to investigate the self-aggregation behavior of the SPS-PEI conjugate. The intensity ratio ( I 339 / I 335 ) of the pyrene excitation spectra as a function of the logarithm of the conjugate concentration of SPS and PEI is trended in Fig. 6 D. The concentration of SPS-PEI at the intersection of the two lines, or CAC value, was 6.46 × 10 − 3 mg/mL. This suggests that the SPS-PEI conjugate has the potential to be used as a hydrophobic drug carrier because it can self-assemble into nanoparticles in an aqueous solution at very low concentrations and maintain the micellar structure before delivering the drug to the intended sites under extremely diluted conditions in the whole blood circulation [ 16 ]. 3.3. Synthesis and characterizations of the siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs To put it succinctly, FA was first created to functionally introduce the carboxylic acid (-COOH), and as Fig. 7 A shows, it was subsequently conjugated with HA by an esterification reaction. The distinctive peak of FA, which can be seen in Fig. 7 C, emerged between 6.6 and 8.6 ppm, indicating that FA was successfully grafted onto HA. In the FT-IR of HA-FA, Fig. 7 B demonstrates that the ester carbonyl group (C = O) stretching vibration peak appeared at 1579 cm − 1 . Additionally, the absorption peaks of the amidogen (-NH 2 ) at 1640 cm − 1 and carboxylic acid (-COOH) at 3280 cm − 1 exceeded the corresponding peak in HA, indicating the synthesis of FA and CD44 receptors double targeting polymer HA-FA. Through electrostatic contact, the cationic SPNPs, HSPNPs, and HFSPNPs can form complexes with siRNA FAM . As illustrated in Fig. 7 D, roughly spherical NPs can be generated by combining the siRNA FAM aqueous solution in the nuclease-free water with the SPNPs, HSPNPs, and HFSPNPs. The physiochemical characteristics of the complexes that formed were modified by adjusting the weight ratios of SPNPs, HSPNPs, and HFSPNPs to siRNA FAM . The resulting siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs complexes exhibit higher zeta potential as the weight ratios increase from 1:1 to 1:20, according to the DLS data in Table 6 . This occurs because more cationic SPNPs, HSPNPs, and HFSPNPs were utilized. The weight ratio of ≥ 0.1 indicated a positive charge for the siRNA FAM /HFSPNPs, siRNA FAM /HSPNPs, and siRNA FAM /SPNPs. The proper particle size of the NPs is also important for efficient siRNA FAM delivery [ 17 ]. When the weight ratio decreases, the siRNA FAM /HFSPNPs, siRNA FAM /HSPNPs, and siRNA FAM /SPNPs develop into larger particles. The 100 ~ 200 nm diameter of the positive surface-charged siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs complexes is appropriate for cell uptake [ 18 , 19 ]. Table 6 Characterizations of blank NPs and siRNA-loaded NPs. Formulation Zeta potential (mV) Size (nm) PDI EE of siRNA (%) SPNPs 23.17 ± 1.63 101.8 ± 9.82 0.207 ± 0.039 - siRNA/SPNPs (1:20) 11.31 ± 1.02 132.2 ± 7.18 0.245 ± 0.028 82.5 ± 6.42 siRNA/SPNPs (1:10) 4.29 ± 0.79 141.7 ± 6.92 0.271 ± 0.019 80.9 ± 7.15 siRNA/SPNPs (1:5) -1.76 ± 0.56 148.6 ± 6.10 0.304 ± 0.032 79.4 ± 3.82 siRNA/SPNPs (1:1) -7.95 ± 0.94 160.8 ± 8.37 0.251 ± 0.027 67.3 ± 5.41 HSPNPs 8.44 ± 1.63 110.9 ± 9.33 0.263 ± 0.036 - siRNA/HSPNPs (1:20) 4.02 ± 0.98 139.3 ± 5.20 0.338 ± 0.031 84.1 ± 6.10 siRNA/HSPNPs (1:10) 1.73 ± 0.31 149.2 ± 6.63 0.372 ± 0.030 79.5 ± 4.87 siRNA/HSPNPs (1:5) -7.82 ± 0.93 157.8 ± 6.10 0.398 ± 0.041 76.2 ± 5.44 siRNA/HSPNPs (1:1) -11.73 ± 1.27 172.2 ± 9.13 0.347 ± 0.034 62.0 ± 3.71 HFSPNPs 10.36 ± 1.73 105.3 ± 4.63 0.252 ± 0.027 - siRNA/HFSPNPs (1:20) 4.67 ± 1.82 137.3 ± 7.10 0.319 ± 0.013 80.9 ± 4.73 siRNA/HFSPNPs (1:10) 1.98 ± 0.83 146.9 ± 8.35 0.379 ± 0.047 73.2 ± 5.02 siRNA/HFSPNPs (1:5) -6.26 ± 0.71 155.5 ± 6.80 0.402 ± 0.020 71.0 ± 4.04 siRNA/HFSPNPs (1:1) -10.49 ± 1.26 176.6 ± 8.23 0.351 ± 0.046 63.2 ± 3.81 3.4. Biocompatibility investigation When it came to the possible uses of nanocarriers in biomedicine, biocompatibility was taken into account. In this case, LO2 and SMMC-7721 cells' cell viabilities were assessed during a 48-hour incubation period with varying quantities of SPNPs, HSPNPs, and HFSPNPs. The outstanding safety of SPNPs, HSPNPs, and HFSPNPs is demonstrated by Fig. 8 C&D, which shows that the cell viabilities of LO2 and SMMC-7721 cells were remained above 85% with concentrations of these substances ranging from 3.1 µg/mL to 100 µg/mL. In addition, since the nanocarriers come into correspondence with blood after being injected intravenously, serum stability is an essential indicator for nanocarriers. Utilizing serum relative turbidity, the stability of HFSPNPs, HSPNPs, and SPNPs was examined. Uncoated SPNPs interacted with serum more over time and had a reversely high positive charge, as seen in Fig. 8 A. On the other hand, following coating, there was a considerable decrease in the interaction between serum and HSPNPs and HFSPNPs, and this interaction did not change much as time passed. Time of the body for the circulation of nanoparticles was prolonged when their binding to non-specific proteins was decreased. Through acid-base titration, the buffer capacities of SPNPs, HSPNPs, and HFSPNPs were determined. The results demonstrate that all of the NPs have a significantly higher proton buffering capacity than NaCl, yet when compared to HFSPNPs, SPNPs and HSPNPs have a lower buffering capacity (Fig. 8 B). Relatively speaking, HSPNPs and SPNPs have less amine groups than HFSPNPs, which could be one explanation. By using the "proton sponge" effect, HFSPNPs can boost the swelling of endocytic vesicles and transport siRNA into the cytoplasm, as demonstrated by this analysis of their strong proton buffering capacity [ 20 ]. 3.5. In vitro drug release study The in vitro drug release characteristics of free siRNA FAM , siRNA FAM /SPNPs, siRNA FAM /HFSPNPs, and siRNA FAM /HSPNPs are shown in Fig. 9 A. The findings showed that, after just 8 h, almost 87% of the free siRNA FAM was released. The release pattern of siRNA/HFSPNPs, on the other hand, was biphasic. A rapid release of 30% siRNA FAM from HFSPNPs happened in the first 8 h, while a slow release of 54.1% took place during the next 64 h. This is most likely due to the fact that the first burst release of siRNA FAM was caused by partially absorbed siRNA on the surface or siRNA FAM entrapped close to the surface of SPNPs, HSPNPs, and HFSPNPs, while the sustained release was caused by the amorphous form of siRNA that was imprisoned inside HFSPNPs [ 21 , 22 ]. The findings suggested that HFSPNPs might find potentially relevance as a siRNA sustained release carrier. 3.6. Study on the pharmacodynamics characteristics of SPNPs, HSPNPs, and HFSPNPs The cellular absorption of various siRNA FAM formulations in SMMC-7721 and LO2 cells was investigated in the present work using flow cytometry and confocal laser scanning microscopy (CLSM). The antagonist of choice was HA and FA owing to its strong affinity for the CD44 and FA receptors. Green fluorescence in Fig. 9 C&D suggested that LO2 and SMMC-7721 cells could receive siRNA FAM -loaded NPs. The time-dependent cellular absorption of siRNA FAM /HFSPNPs, as depicted in Fig. 9 B, suggested that the NPs accumulated inside cells gradually to exert their therapeutic effects. Similar results were also observed in SMMC-7721 cells. More importantly, the higher accumulation efficiency of siRNA FAM /HFSPNPs compared with siRNA FAM /SPNPs and siRNA FAM /HSPNPs might be ascribed to the high affinity of the HFSPNPs for CD44 and FA. Furthermore, LO2 and SMMC-7721 cells presented a greater decrease in green fluorescence intensity with the prior addition of HA and FA. This was because HA (or FA) could competitively bind to CD44 (or FA) and restrain the endocytosis of CD44 (or FA) [ 23 ]. As predicted, after 4 h of incubation with siRNA FAM /SPNPs, siRNA FAM /HSPNPs, and siRNA FAM /HFSPNPs, the green fluorescence intensities in SMMC-7721 cells were stronger than those in LO2 cells, which somewhat supports CD44 (or FA) -mediated endocytosis. Quantitative flow cytometry tests were carried out to further validate the liver-targeting capacity of SPNPs, HSPNPs, and HFSPNPs. The uptake of each siRNA FAM formulation in SMMC-7721 cells was time-dependent, as predicted, and at each time point, the mean fluorescence intensity of the siRNA FAM /HFSPNPs group was significantly higher than that of the other groups (Fig. 9 E). These outcomes agreed with the CLSM findings. After confirming the high siRNA uptake utilizing NPs, we employed siBMP7 to assess the effectiveness of gene silencing in SMMC-7721 cells via western blot analysis. The silencing efficacy varies based on the weight ratios, as Fig. 9 F&G illustrating. The CLSM and flow cytometry showed that as the weight ratios of siBMP7 to HFSPNPs growing, more siBMP7 was internalized into the cells, resulting in a drop in BMP7 levels in the cells. In comparison to the control group, the siBMP7/HFSPNPs with a weight ratio of 20:1 could lower the BMP7 expression to about 20%. Based on the in vitro experiment's poor absorption capacity, we eliminated the group with free siRNA FAM from the in vivo trial. The efficacy of enhancing site-specific delivery of siRNA and HCC prevention was compared among control, siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs groups. In the in vivo model, SMMC-7721 cells were sequentially pretreated for 7 days in all animals except those in the control group. After that, the model was constructed with three distinct formulations and saline as a control. Figure 10 A&B showed the differences in body weights and tumor volumes. Each carried the same body weight for the duration that the tumor was borne. All body weights, with the exception of the control group, began to develop less quickly after the 7th day of formulation administration. Tumor volume remained same on day 7 but altered on day 9, while siBMP7/HFSPNPs showed total tumor inhibition before the experiment ended, indicating that treatment with siBMP7/HFSPNPs might more successfully inhibit tumor growth. Ultimately, the siBMP7/HFSPNPs inhibition rate may reach 69.5% based on the tumor quality displayed in Fig. 10 C&D. To bolster the protective impact of siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs, histological studies were conducted. As demonstrated by H&E staining of tumor sections, different formulations reduced severe lesions, with the siBMP7/HFSPNPs group showing the largest preventative advantages (Fig. 11 A). As seen in Fig. 11 B, more cells were participating in the autophagic process when treated with siBMP7/HFSPNPs as compared to cells treated with siBMP7/SPNPs and siBMP7/HSPNPs. These cells also displayed greater apoptosis and stronger red fluorescence spots. The results imply that the HA-FA coating can promote carrier uptake by SMMC-7721 cells and thus trigger the demise of cancerous cells. Additionally, from a pharmacological perspective, western blot might show that HFSPNPs can mute the BMP7 gene more successfully, enabling increased siBMP7 distribution to cells through Fig. 11 C&D. 3.7. Safety of nanoparticles in vivo As shown in Fig. 12 , several NPs formulations demonstrate remarkably in vivo safety. Tumor-bearing mice treated with siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs showed no significant injury to the kidney, liver, spleen, lung, or heart aside from the tumor tissue. 4. Discussion Natural polysaccharides are superior to other non-viral vectors for gene delivery for the reason they are simple to prepare and produce, exhibit biological activity such as the ability to identify liver cells and macrophages, and are easily hydrolyzed and enzymolized in cells. The breakdown products of monosaccharides and amino compounds also have low toxicity and may even provide nutrients and promote cell growth. Polysaccharide recognition by cell surface receptors could speed up cell internalization after several naturally occurring polysaccharides in the amination reaction were designed and synthesized using the gene carrier with high transfection efficiency. This represents the establishment of a cationic polysaccharide carrier with low cytotoxicity and immunogenicity, good biodegradability, and high solubility in water, compared to those synthesized using other cationic polymers [ 24 ]. Importantly, whereas certain polysaccharides have helical structures, they can hydrogen link with polynucleotide polymers to generate a hybrid three-strand helical polymer that can effectively prevent polynucleotide degradation [ 25 ]. Naturally occurring polysaccharides have low toxicity and good biocompatibility, while PEI, a traditional transfection reagent, has a high transfection rate and significant cytotoxicity. Due to this, scientists combined the benefits of the two materials by modifying natural polysaccharides using PEI as the gene carrier [ 26 ]. Since HA is an acidic mucopolysaccharide [ 27 ], it can be chemically or physically changed to create derivatives that have a variety of uses as materials for drug delivery vehicles. HA and its derivatives have found widespread use as drug carrier materials because of their exceptional biocompatibility, biodegradability, receptor binding, non-toxicity, and immunogenicity. Human cells have a high number of HA receptors. HA and its derivatives can target specific cell surface receptors to deliver medicines to those cells. Many receptors are under investigation, such as the HA receptor-1 on lymphatic endothelial cells, the CD44 receptor, the HA-mediated motivation (RHAMM) receptor, and the HA endocytosis receptor [ 28 , 29 ]. Many tumor cells have overexpressed CD44 and RHAMM receptors on their surface, which when paired with HA-modified vectors allows for active drug vector targeting. An amphiphilic HA-deoxy acid linking self-assembly of redox-sensitive micelles, for instance, was created by Li et al. to deliver paclitaxel specifically into cells [ 30 ]. The glycoprotein known as the FA receptor is expressed at low to moderate levels in the lungs, kidneys, and choroids but is virtually absent from other normal tissues. In the majority of malignant tumors, including ovarian, cervical, kidney, breast, and colon cancers, the high-affinity FA receptor was significantly expressed. As a naturally occurring ligand of the FA receptor, FA is currently inexpensive, non-toxic, and simple to access and alter. In addition, it displays excellent biocompatibility, robust FA receptor binding, and minimal immunogenicity. Additionally, the FA molecule is stable and may sustain a long blood circulation or storage period while maintaining a reasonably high receptor affinity. Direct coupling of FA to drug molecules and coupling of FA on the surface of the drug carrier are the two main methods used by the FA receptor-mediated active targeted drug delivery system to target tumor locations. The solubility of commonly used anti-tumor drugs is normally lower in water under physiological settings. This is due to the direct coupling of FA to the drug molecules, which further reduces the solubility of the medication in water and ultimately reduces its bioavailability. Therefore, the coupling of FA to the surface of a drug carrier strategy has research value; currently, the FA receptor is the main focus in research on the active targeting drug delivery system. To sum up, the research successfully created self-assembled nanoparticles. HFSPNPs could effectively accumulate in hepatic cells by HA-FA-mediated uptake, according to studies done both in vitro and in vivo. This suggests that HFSPNPs could be a viable delivery mechanism for drugs to be delivered to the liver, where they might exhibit their protective effects. In SMMC-7721 cells, the siBMP-7/HFSPNPS particles showed strong BMP7 knockdown along with strong in vitro and in vivo stability, strong gene loading capacity, high cell uptake, transfection efficiency, and tumor-targeting properties. Even though no harm was done to other organs, the tissue distribution staining experiment demonstrated that HFSPNPs may increase the amount of siBMP7 that accumulates in the tumor. The release of siBMP7 from the nanoparticles allowed for the achievement of the gene therapy goal by promoting apoptosis in cancer cells and reducing the expression of BMP7 protein in tumor tissue cells. These findings suggest that HFSPNPs may be used as an HCC tumor-targeting drug in the treatment of HCC, which has implications for more research. Declarations Acknowledgments We would like to thank Editage (www.editage.cn) for their assistance with English language editing. Ethical Approval This study was approved by the Laboratory Animal Center, Heilongjiang University of Chinese Medicine and also followed the ARRIVE guidelines. Competing interests The authors report there are no competing interests to declare. Data availability The figures and tables supporting the results of this study are included in the article, and the original datasets are available from the first author or corresponding author upon request. Funding This work was supported by the National Natural Science Foundation of China under Grant number 81603418; and the Central Government Supports Local College Reform Projects under Grant number 2020YQ05. The funding agencies had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Authors' contributions Haotian Bai: Writing the original manuscript draft; Conceptualization; Data curation; Visualization. Jing Yang: Supervision; Resources; Funding acquisition. Junhao Zhang: Data validation; Software; Data curation Rui Wang: Formal analysis; Methodology; Project administration. Authors' information Haotian Bai (ORCiD ID: 0000-0002-7158-7668): College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email: [email protected] Jing Yang (ORCiD ID: 0000-0002-9705-3883): College of Basic Medical Science, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email: [email protected] Junhao Zhang: College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email: [email protected] Rui Wang (ORCiD ID: 0000-0003-4770-3515): College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China; College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China; Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine, Ministry of Education, Harbin, Heilongjiang 150040, China. Email: [email protected] References L.A. Torre, F. Bray, R.L. Siegel, et al., Global cancer statistics, 2012. CA Cancer J Clin. 65 (2015) 87–108. D.H. Kim, J.J. Ross, Strategies for silencing human disease using RNA interference. Nat Rev Genet. 8 (2007) 173–184. W. Zhao, J. Zhao, M.C. Yong, et al., Self-healing polysaccharide-based hydrogels as injectable carriers for neural stem cells. Sci Rep. 6 (2016) 37841–37852. K. Wong, G. Sun, X. 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Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.tif SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6119667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427854972,"identity":"8224976e-41d2-4e2c-abd2-9b241358dd4c","order_by":0,"name":"Haotian Bai","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haotian","middleName":"","lastName":"Bai","suffix":""},{"id":427854973,"identity":"fed36c47-7d11-49fb-be37-409557ab9b7d","order_by":1,"name":"Jing Yang","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Yang","suffix":""},{"id":427854974,"identity":"f541cfc0-015b-4fce-ad90-786c1d2f3b38","order_by":2,"name":"Junhao Zhang","email":"","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junhao","middleName":"","lastName":"Zhang","suffix":""},{"id":427854975,"identity":"601fbc96-841f-4d39-a5f4-a4c7fb7b0e23","order_by":3,"name":"Rui Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAlElEQVRIiWNgGAWjYHACA4YPDBIkamGcQbIWZh7S1N8+vE3a5o9FHn8D88NHN4jSci6t2Di3TaJY4gCbsXEOMVrMzvAYPs5tkEhsOMDDJk2sFoPDFn8kEueTosXwMQObROIGorXYn2ErNuxtk0jceJhYv0j2MG+T+PGnLnHe8eaHj4nSggDMpCkfBaNgFIyCUYAPAACzUSzdqcUp/AAAAABJRU5ErkJggg==","orcid":"","institution":"Heilongjiang University of Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Rui","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-02-27 09:38:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6119667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6119667/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78657694,"identity":"8478361d-1ac2-4ad6-9a0a-0b8836d5ddf6","added_by":"auto","created_at":"2025-03-17 09:39:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1284271,"visible":true,"origin":"","legend":"\u003cp\u003eSafflower polysaccharide (SPS) separation and purification process.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/a26b37d9bb00ef43629f3872.png"},{"id":78657696,"identity":"622a8640-b697-48c1-b8d5-ddb4447ecd28","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3284976,"visible":true,"origin":"","legend":"\u003cp\u003eThe production process of HA-SPS-PEI and HA-FA-SPS-PEI.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/eace3095efbb4e4ba99ada1e.png"},{"id":78658335,"identity":"3f2aab77-918a-47e2-8cdb-292c0c14ce43","added_by":"auto","created_at":"2025-03-17 09:47:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1661885,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the tumor-bearing model.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/e9ce6cac7581ab315bf27db7.png"},{"id":78657703,"identity":"e94fbcc2-5fdf-4140-8855-db2974512c71","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1426662,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Elution curve of the crude polysaccharides (SPS) obtained from Safflower on a column of DEAE-cellulose. The fractions containing the polysaccharides were pooled and named as SPS-a (distilled water) to SPS-b (0.25 M NaCl), respectively. (B) Elution curve of one major fraction SPS-1 on a column of SephadexG-100. (C) Elution curve of one major fraction SPS-2 on a column of SephadexG-100.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/9fc4167da066c6f377819ed4.png"},{"id":78658338,"identity":"06021e99-37ed-4eba-a195-cc25ef094d4e","added_by":"auto","created_at":"2025-03-17 09:47:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5492716,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Standard curve of glucose and uronic acid. (B) Ultraviolet spectrum of SPS-1 and SPS-2. (C) HPLC chromatograms of PMP derivatives of 7 standard monosaccharides and monosaccharides of SPS-1 and SPS-2. (D) (a) HPGPC profile of SPS-1 and SPS-2. (b) The calibration curve of Dextran standards with different molecular weight. (E) Congo Red structure analysis of SPS-1 and SPS-2. (F) The infrared measurement of SPS-1 and SPS-2. (G) \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum of the SPS-1 and SPS-2. (H) Standard curve of KIO\u003csub\u003e4.\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/9a8a70df8c3ca5ce04039bae.png"},{"id":78658340,"identity":"270e5c23-93e7-4b0d-9f1d-3fa1034faaba","added_by":"auto","created_at":"2025-03-17 09:47:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2360813,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterizations of SPS-PEI conjugate. (A) The synthesis of SPS-PEI. (B) FT-IR spectra of SPS, SPS-PEI in DMSO-d6. (C) \u003csup\u003e1\u003c/sup\u003eH-NMR spectra of SPS, SPS-PEI. (D) CAC of SPS-PEI conjugate.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/dc1f941469e928984a3c0b71.png"},{"id":78657708,"identity":"a015dd31-8a70-4599-84dc-88712f8abbbd","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6280921,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterizations of HA-FA conjugate and self-assembled nanoparticles. (A) The synthesis of HA-FA. (B) FT-IR spectra of HA-FA. (C) \u003csup\u003e1\u003c/sup\u003eH-NMR spectra of HA-FA. (D) TEM images of SPNPs, HSPNPs, and HFSPNPs.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/f62ed0ade78fafceb54a2a48.png"},{"id":78657693,"identity":"4ac332e2-dff6-4e17-850a-f81363d9c96e","added_by":"auto","created_at":"2025-03-17 09:39:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2230087,"visible":true,"origin":"","legend":"\u003cp\u003eBiocompatibility of nanoparticles. (A) Serum stability of SPNPs, HSPNPs, and HFSPNPs. (B) Acid-base titration curves of NaCl, SPNPs, HSPNPs, HFSPNPs, and PEI. (C) Cytotoxicity analysis of SPNPs, HSPNPs and HFSPNPs after incubation for 96 h on LO2 cells. (D) and SMMC-7721 cells.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/8b214473e7e09dd7ea9f54d7.png"},{"id":78657735,"identity":"3ce1b2fe-ef57-4d8f-8a14-a6c8b47ff074","added_by":"auto","created_at":"2025-03-17 09:39:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4690985,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro drug release (A) and cellular uptake behavior of free siRNA and different nanoparticles. (B) CLMS images of LO2 cells incubated with siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs at different time points. (C) CLMS images of LO2 cells (D) and SMMC-7721 cells after 4 h treatment with different formulations. (E) Quantitative flow cytometry analysis of the different formulations in SMMC-7721 cells at different time points. Values are represented by the mean ± standard deviation, n = 3. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01,\u003csup\u003e ***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/bcc4d138b65a81370600e51a.png"},{"id":78657724,"identity":"d04cecc2-612d-4b15-9b57-ccf1f37d418c","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":6118168,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo study of different nanoparticles acting on HCC tumors. (A) Changes in body weight of mice. (B) Changes in tumor volume of mice. (C) Schematic diagram of tumor in vitro (D) Weight of separated tissue.\u003c/p\u003e","description":"","filename":"Figure101.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/7411725c07f4486cd06672b3.png"},{"id":78657723,"identity":"5bd61ce8-3217-4d1e-9251-6d49e2699042","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":16211897,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo BMP7 expression and tumor treatment. (A) H\u0026amp;E staining of HCC tumor tissues after respective treatments (Scale bars = 100 μm). (B) AO staining of tumor supernatant after respective treatments (Scale bars = 200 μm). (C) MMP-9 protein bands. (D) and levels by western blotting.\u003c/p\u003e","description":"","filename":"Figure111.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/2213ec925bea9b44bb67db3a.png"},{"id":78657732,"identity":"64a1cae7-fff2-4636-8c95-a8949b6a60c3","added_by":"auto","created_at":"2025-03-17 09:39:29","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":10274035,"visible":true,"origin":"","legend":"\u003cp\u003eH\u0026amp;E staining of major organs after respective treatments (Scale bars = 100 μm).\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/609c10749f1672acba966315.png"},{"id":81679786,"identity":"bcd063c9-0345-4730-8ad4-8ebc3dbb0406","added_by":"auto","created_at":"2025-04-30 08:47:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":56872709,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/da876295-77ae-450a-ac01-7b9662960975.pdf"},{"id":78657698,"identity":"a3a1abd7-b344-4636-ac3d-6112cf4f51f4","added_by":"auto","created_at":"2025-03-17 09:39:28","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8842268,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/7da4c1bd5bf026d85a80cd38.tif"},{"id":78660231,"identity":"427f5175-b43c-4b70-9eca-431e0853f6f7","added_by":"auto","created_at":"2025-03-17 10:03:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3959991,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6119667/v1/7a6bbacf83173126b2835131.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eSynthesis, characterization, and application of self-assembled safflower polysaccharide nanoparticles as liver targeting drug delivery carrier\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC), one of the most widespread malignancies with the highest prevalence, is the second most common cause of cancer-related death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Chemotherapy and traditional surgery have serious side effects and do not produce the best outcomes. In recent years, RNA interference (RNAi) technology has become a popular gene interference technique and an emerging cancer treatment approach in addition to surgery, chemotherapy, and radiation therapy. Strong selectivity, good specificity, quick action, and high gene silencing efficiency are all advantages of using RNAi technology for the treatment of hepatocellular carcinoma. This method may be used to decrease the expression of some crucial oncogenes and key signaling pathway molecules in tumor cells, thereby preventing the growth of tumors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearchers' interest in natural polysaccharides, a secure, effective, and low-toxic gene carrier, has gradually grown. After proteins and nucleic acid, polysaccharides are the third type of macromolecule that stores biological information. Natural polysaccharides continue to garner interest in the realm of biological materials due to their high non-toxicity, good biocompatibility, and biodegradability [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Safflower polysaccharide (SPS) is a naturally occurring polysaccharide obtained from Compositae \u003cem\u003eCarthamus tinctorius L.\u003c/em\u003e It has anti-tumor, antioxidant, and immunity-boosting characteristics and also contains the active group -OH. The synthesis of SPS with various biological effects can therefore be made possible by the chemical alteration of -OH. Polyethyleneimine (PEI) is a classical transfection agent with a high transfection rate, exhibiting good biocompatibility and low toxicity. Owing to their joint advantages, researchers used the two materials to modify the natural polysaccharide with PEI as the gene carrier [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cell internalization may be accelerated by identifying polysaccharide receptors on the cell surface. Cationic polysaccharide carriers have advantages over other cationic polymers concerning low cytotoxicity and immunogenicity, good biodegradability, and high water solubility [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe receptor-mediated targeted gene delivery system is the most advanced cell-specific vector system with the advantages of high specificity and high affinity, and it greatly improve the drug delivery efficiency of drugs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The commonly used specific receptors include hyaluronic acid (HA), folic acid (FA), integrin, and galactose receptors. As a new targeted drug delivery strategy, dual-receptor-mediated targeted drugs could deliver nanoparticles to specific sites more effectively. Therefore, the dual-receptor rake strategy has been widely used in drug delivery systems in recent years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBone morphogenetic proteins (BMPs) are a group of functional proteins that are widely involved in biological processes that regulate the proliferation, differentiation, and apoptosis of various cells. BMP expression is involved in the formation of embryos and organs, as well as in the formation and development of tumor diseases. BMP7 is a member of the transforming growth factor beta (TGF-β) superfamily. An increasing number of studies have shown that BMP7 is significantly expressed in various epithelial tumors, such as breast cancer, primary melanoma, prostate cancer, and colorectal cancer [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the relationship between BMP7 gene changes and malignant biological characteristics of hepatocellular carcinomas, such as malignant differentiation degree, proliferation, invasion, metastasis, and recurrence, is rarely reported in local and international research.\u003c/p\u003e \u003cp\u003eBased on the above studies, cationic safflower polysaccharide (SPS-PEI) was selected as the basic gene carrier in this study, and siRNA was bound by electrostatic adsorption. HA and FA were covalently combined to form a polymer through esterification reaction, which was then coated with SPS-PEI to improve delivery efficiency. While shielding the positive charge to reduce cytotoxicity, FA and HA receptors (CD44) on SMMC-7721 cell surfaces could be simultaneously targeted to construct a double-targeted nano gene vector.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eThe plant material collected was identified as the dried tubular flower of the Compositae \u003cem\u003eC. tinctorius L.\u003c/em\u003e by Professor Rui Wang (College of Pharmacy, Heilongjiang University of Chinese Medicine) at the Medical Botany Center of Heilongjiang University of Chinese Medicine, where a voucher specimen (No. 20210493) has been deposited.\u003c/p\u003e \u003cp\u003ePolyethyleneimine (PEI), N, N-carbonyl imidazole (CDI), tetramethylsilane (TMS), folic acid (FA), hyaluronic acid (HA), N-butyl hydroxy (NHS), dicyclohexyl-carbodiimide (DCC), and anti-fluorescence quenching (AFQ) were obtained from Shanghai Aladdin Biochemical Technology Co. Ltd. (Shanghai, China). 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Annexin V-FITC/PI double staining kit, AO Staining, Hoechst33258 and protein marker were obtained from Sangon Biology Co. Ltd. (Shanghai, China). BMP-7, β-actin, and Goat anti-rabbit Immunoglobulin G (IgG) (H\u0026amp;L) were purchased from Abways Company (Shanghai, China). Detailed information about siRNA can be found in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. SiRNA\u003csup\u003eFAM\u003c/sup\u003e was synthesized by Suzhou Jima Gene Co. Ltd. (Suzhou, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSequence of siRNA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUpstream primers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDownstream primers\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiBMP-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCG UCC UCU ACU UCG AUG AdTdT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUCA UCG AAG UAG AGG ACG GdTdT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA\u003csup\u003eFAM\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUUC UCC GAA CGU GUC ACG UdTdT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACG UGA CAC GUU CGG AGA AdTdT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTC CAT CCT GGC CTC GCT GT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCT GTC ACC TTC ACC GTT C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell lines and cell culture\u003c/h2\u003e \u003cp\u003eThe human hepatocellular carcinoma cell (SMMC-7721) and the normal human hepatic cell (LO2) were obtained from Shanghai Fuheng Biotechnology Co. Ltd. (Shanghai, China). SMMC-7721 cells and LO2 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Vivacell, Shanghai, China) supplemented with 10% (v/v) fetal bovine serum (Hyclone) and antibiotics (100 U/ mL penicillin and 100 U/mL streptomycin) (Hyclone) at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Animals\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice that were specifically pathogen-free (SPF) and weighed (20\u0026thinsp;\u0026plusmn;\u0026thinsp;2) g were purchased from Liaoning Changsheng Biotechnology Co. Ltd. (Shenyang, China).The mice were housed in polypropylene cages at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C at a relative humidity of 50\u0026thinsp;\u0026plusmn;\u0026thinsp;20% and a 12-h light-dark cycle with free access to food and water. The study was approved by Ethics Committee of Heilongjiang University of Chinese Medicine (approval number:202208011). All methods were carried out in accordance with relevant guidelines and regulations. This study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Separation and purification of safflower polysaccharides\u003c/h2\u003e \u003cp\u003eThe unrefined SPS was extracted according to a method used in a previous study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The specific operational details are presented in Supplement 1. The phenol-sulfuric acid method was used for color tracking, and the absorbance value was measured at 490 nm. Polysaccharide samples with the same elution peak were collected. The elution curve was plotted using the tube number as abscissa (\u003cem\u003eX\u003c/em\u003e) and absorbance (A) as ordinate (\u003cem\u003eY\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eComponents were separated and purified form Sephadex G-100 and prepared into a 1 mg/mL solution after dialysis, concentration, and freeze drying. Afterwards, the solution was scanned using the UV spectrophotometer within the wavelength range of 200～800 nm. Distilled water was used as the blank control. The characteristic absorption peaks of nucleic acid (260 nm) and protein (280 nm) were detected. The separation and purification flow chart is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Structural identification of safflower polysaccharide\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Determination of total carbohydrate and uronic acids\u003c/h2\u003e \u003cp\u003eGlucose and glucuronic acid control solutions with concentrations of 20, 40, 60, 80, and 100 \u0026micro;g/mL were prepared. Standard curves were drawn using glucose concentration as abscissa (\u003cem\u003eX\u003c/em\u003e) and absorbance as ordinate (\u003cem\u003eY\u003c/em\u003e). SPS-1 and SPS-2 were accurately weighed to prepare their respective 100 \u0026micro;g/mL aqueous solutions. Total polysaccharides and uronic acids were determined using sulfuric acid-anthracone and sulfuric acid-carbazole method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Determination of relative molecular mass\u003c/h2\u003e \u003cp\u003eThe relative molecular weights (4600～133800) of the dexglucoside series of reference substances were added to distilled water to prepare a 1 mg/mL solution, and then filtered using a 0.45 \u0026micro;m microporous membrane. Then 500 \u0026micro;L of each of the aforementioned reference solution was taken in turns, starting from small to large molecules. Three replicates of each sample were taken in parallel, and the retention time of their elution peaks were recorded under the known chromatographic conditions. The standard curve was derived from the logarithm of the molecular weight (M\u003csub\u003eW\u003c/sub\u003e) of the reference substance and its corresponding retention time (tR), and the linear regression equation was lgM\u003csub\u003eW\u003c/sub\u003e = a + btR. SPS-1 and SPS-2 prepared using the aforementioned method were treated with 50 \u0026micro;L and determined according to the same method, and the retention time (tR) of the polysaccharide was recorded. The weight average molecular weight, Mw, number average molecular weight, Mn, and polydispersion index, D, of polysaccharides were calculated according to the known standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Analysis of monosaccharide composition\u003c/h2\u003e \u003cp\u003eThe monosaccharide components were analyzed using pre-column derivatization high performance liquid chromatography (HPLC). The purified 50 mg SPS-1 and SPS-2 were respectively weighed in a hydrolysis tube, and 1 mL water and 1 mL 4 mol/L trifluoroacetic acid were added. Under conditions of nitrogen protection, the water was hydrolyzed at 110\u0026deg;C for 2 h and then cooled to room temperature. Then 0.1 mL was placed in a 4 mL centrifuge tube, and vacuum dried at 60\u0026deg;C for 2 h. The standard monosaccharides were obtained by sequentially adding 5 mg of D-glucose, D-galactose, D-galacturonic acid, D-xylose, D-mannose, L-rhamnose, and L-arabinose to a 25 mL volumetric flask. In addition, 500 \u0026micro;L of the standard monosaccharide solution and 2 hydrolyzed polysaccharide samples were placed in a centrifuge tube. Then 0.05 mL 0.3 mol/L NaOH and 0.05 ml PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution were added to each tube. The reaction was performed in a water bath at 70\u0026deg;C for 60 min under nitrogen protection and cooled to room temperature. Then 0.5 mL 0.3 mol/L HCl, 0.75 mL H\u003csub\u003e2\u003c/sub\u003eO, and 1.5 mL chloroform were added for extraction. The chloroform layer was discarded and extracted three times. The water layer was combined with PMP-derived products on the upper layer and filtered by means of a 0.45 \u0026micro;m filter membrane. The samples were directly injected for gas chromatography (GC) analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. FT-IR analysis\u003c/h2\u003e \u003cp\u003eIn order to perform the FT-IR study, 3 mg of SPS-1 and SPS-2 were ground and compressed using KBr. A total of 24 scans were performed within the 4000～400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5. \u003csup\u003e1\u003c/sup\u003eH-NMR analysis\u003c/h2\u003e \u003cp\u003ePrior to NMR analysis, 30 mg of over-dried polysaccharide was dissolved in 0.5 ml of 99.97% D\u003csub\u003e2\u003c/sub\u003eO for 6 hours after being repeatedly freeze-dried in D\u003csub\u003e2\u003c/sub\u003eO to exchange deuterium. On a Bruker AV-500 spectrometer (Bruker, Rheinstetten, Germany), the 1H- was acquired at 30\u0026deg;C at 500 MHz for \u003csup\u003e1\u003c/sup\u003eH. The acquisition time (AT\u0026thinsp;=\u0026thinsp;0.5 s), sweep width (SWH\u0026thinsp;=\u0026thinsp;20,000 Hz), relaxation delay (d1\u0026thinsp;=\u0026thinsp;10 s), and 90\u0026deg; pulse time (p1\u0026thinsp;=\u0026thinsp;2.5 \u0026micro;s) were the parameters that were specified for the \u003csup\u003e1\u003c/sup\u003eH-NMR spectra. The recordings were made with a relaxation delay of 1.5 s. Chemical shifts are measured in parts per million (ppm) and are calibrated internally using acetone, which has a δ31.21 ppm for carbon and a δ2.19 ppm for hydrogen. The TopSpinTM3.5 program from Bruker was used to evaluate the data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6. Structure analysis of Congo red\u003c/h2\u003e \u003cp\u003eSolutions of Congo red (0.2 mmol/L) and SPS-1/2 (2 mg/mL) were made. After thoroughly mixing 1.5 mL of Congo red solution with 1 mL of polysaccharide solution and 0.5 mL of water, the mixture was split into 10 equal portions. Concurrently, the aqueous solution containing polysaccharides was converted to an aqueous solution and designated as the Congo red blank control group. For around ten minutes, the solution was incubated at room temperature. At a wavelength of 200\u0026thinsp;~\u0026thinsp;600 nm, UV scanning was carried out, and the highest absorption wavelength was noted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.5.7. Periodic acid oxidation experiment\u003c/h2\u003e \u003cp\u003eFirst, a consistent volume of 15 mmol/L potassium periodate (KIO\u003csub\u003e4\u003c/sub\u003e) original solution was created, and its concentration was diluted to 10, 15, 30, 45, and 60 \u0026micro;mol/L KIO\u003csub\u003e4\u003c/sub\u003e solution. The UV absorbance was then measured at 223 nm. KIO\u003csub\u003e4\u003c/sub\u003e concentration was represented by the x-coordinate (\u003cem\u003eX\u003c/em\u003e) on the standard curve, and absorbance value by the y-coordinate (\u003cem\u003eY\u003c/em\u003e). To stop the light reaction at room temperature, an additional 5 mg of SPS was weighed and 10 mL of KIO\u003csub\u003e4\u003c/sub\u003e solution (15 mmol/L) was added. Subsequently, a volume of 25 mL was maintained while 100 \u0026micro;L was taken at 0, 4, 8, 12, 24, and 48 h. The absorbance value at 223 nm was obtained using distilled water as the blank control. The absorbance value steadily dropped as KIO\u003csub\u003e4\u003c/sub\u003e was used up until the measured value steadied, signifying that the reaction was finished and allowing for the calculation of KIO\u003csub\u003e4\u003c/sub\u003e consumption. In order to use up the extra KIO\u003csub\u003e4\u003c/sub\u003e, 1 mL of glycol was added last, and the reaction was stopped by shaking for 10 minutes. To ascertain the amount of formic acid produced, 2 mL of the previously described oxidized solution was added along with 2 drops of phenolphthalein indicator, and the mixture was titrated with 0.01 mol/L NaOH.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Synthesis and characterizations of the SPS-PEI conjugate\u003c/h2\u003e \u003cp\u003eAs per an earlier study, the SPS was isolated from the dry safflower flowers and refined. An esterification reaction could bond PEI to SPS [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The specific operational details are presented in Supplement 2. Using DMSO d6 as the solvent at 400 MHz, FT-IR spectroscopy Nicolet 170SX and \u003csup\u003e1\u003c/sup\u003eH-NMR spectrometry were used to determine the chemical structures of SPS and SPS-PEI.\u003c/p\u003e \u003cp\u003eBy employing fluorescence spectroscopy with pyrene as a hydrophobic probe, the critical aggregation concentration (CAC) of the SPS-PEI conjugate was used to measure its self-aggregation characteristic. In summary, a succession of volumetric flasks were filled with a known quantity of pyrene in acetone, and the acetone was blown out using N\u003csub\u003e2\u003c/sub\u003e. Following the formation of a pyrene film, different concentrations of the SPS-PEI conjugate solution were made and put into each volumetric flask, yielding a final solution with a pyrene concentration of 6 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e M. To acclimate the pyrene and conjugate, the mixture was shaken for 12 h at 37\u0026deg;C in a thermostatic shaking water bath. With an emission wavelength of 372 nm, the fluorescence excitation spectra were obtained using a F7000 fluorescence spectrometer operating in the 300\u0026thinsp;~\u0026thinsp;360 nm range. For the purpose of measuring the intensity ratio, the fluorescence intensities (I\u003csub\u003e339\u003c/sub\u003e and \u003cem\u003eI\u003c/em\u003e\u003csub\u003e335\u003c/sub\u003e) at 339 and 335 nm were noted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Synthesis and characterizations of the siRNA/SPNPs, siRNA/HSPNPs, and siRNA/HFSPNPs\u003c/h2\u003e \u003cp\u003eAn esterification reaction could bind HA to FA. In short, 1.0 g DCC and 288 mg NHS were dropped and activated at 30\u0026deg;C for 5 h after 50 mg FA was dissolved in 3 mL DMSO at room temperature for 30 min. The activation process produced precipitation, which was filtered out. Moreover, 10 mL of formamide containing 100 mg of HA was heated to 40\u0026deg;C to fully dissolve it, and the mixture was added drop by drop over the course of 48 h [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Filtration was used to remove the precipitate, and for 48 h, light protection was used while dialyzing with deionized water. The HA-FA conjugate was obtained by lyophilizing the water, which was replaced every 6 h. The FT-IR and \u003csup\u003e1\u003c/sup\u003eH-NMR analysis procedure used for HA-FA was the same as that used for SPS. HA-SPS-PEI and HA-FA-SPS-PEI were produced using the magnetic-stirring technique depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In short, SPS-PEI was gradually added to the aqueous solutions of HA and HA-FA that were magnetically agitated and set at 1 mg/mL for a duration of 15 min. Thus, HA-SPS-PEI and HA-FA-SPS-PEI were acquired.\u003c/p\u003e \u003cp\u003eThe dialysis-sonication approach was used to prepare SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (SPNPs, HSPNPs, and HFSPNPs). In summary, 10 mg of SPS-PEI, HA-SPS-PEI, or HA-FA-SPS-PEI was dissolved in 1 mL of DMSO and then gradually added to 3 mL of deionized water while being continuously stirred at room temperature (1000 rpm). The combination was dialyzed against deionized water for a day. A well-dispersed nanoparticle solution was then achieved by sonicating the mixture for 3 min. Zeta potentials of complexes produced at a mass concentration of 1 mg/mL and dissolved in an appropriate PBS solution were measured using a Nano90 Malvin Laser at 25\u0026deg;C and a 90\u0026deg; scattering angle using dynamic light scattering (DLS).\u003c/p\u003e \u003cp\u003eWith the exception of adding various quantities of siRNA\u003csup\u003eFAM\u003c/sup\u003e to the SPS-PEI DMSO solution, the same protocol was used to generate the siRNA\u003csup\u003eFAM\u003c/sup\u003e-loaded SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (siRNA/SPNPs, siRNA/HSPNPs, and siRNA/HFSPNPs). Centrifugation was used to extract free siRNA for 15 min at 3000 rpm. After lyophilization, siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs were finally produced.\u003c/p\u003e \u003cp\u003eIn order to assess the loading of siRNA in SPNPs, HSPNPs, and HFSPNPs, a predetermined volume of lyophilized siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/ HFSPNPs was dissolved in an appropriate PBS solution. The fluorescence intensity value was then measured by adding Ribogreen fluorochrome (x200) and TBE solution. With the assistance of a spectrofluorometer set to measure at 436 nm, the siRNA\u003csup\u003eFAM\u003c/sup\u003e encapsulation efficiencies (EE) of SPNPs, HSPNPs, and HFSPNPs were computed as follows: EE (%)=\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{C}\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}-\\text{C}free}{Ctotal}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs in PBS solution (pH 7.4) at 1 mg/mL were subjected to DLS analysis at 25\u0026deg;C using a Nano 90 Malvern Laser to determine their mean particle size, polydispersity index (PDI), and zeta potential. In order to investigate the morphology of the nanoparticles in more detail, the sample solutions (1 mg/mL) were placed onto copper grids covered with carbon, negatively stained for 2.5 min with a solution of 1% phosphotungstic acid, and allowed to air dry at room temperature. An H-7000FA Transmission Electron Microscope was used to image the nanoparticles' ultrastructure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Biocompatibility investigation of blank nanoparticles\u003c/h2\u003e \u003cp\u003eIndicators for assessing the biocompatibility of the SPNPs, HSPNPs, and HFSPNPs included serum stability, cytotoxicity, and buffer capacity.\u003c/p\u003e \u003cp\u003eFor the serum stability test, 100 \u0026micro;L of FBS was combined with 0.9% NaCl (negative group) or the same volume of SPNPs, HSPNPs, and HFSPNPs solution at different concentrations. A UV-Vis spectrophotometer was used to measure the absorbance of each group at 630 nm at 0, 2, 6, 12, 18, and 24 h after incubation in a water bath at 37\u0026deg;C away from light. By computing the absorbance ratio between the samples and the negative group at each time point, the relative turbidity was determined.\u003c/p\u003e \u003cp\u003eAfter being seeded at a density of 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates, LO2 and SMMC-7721 cells were cultivated for a whole night. Upon being treated for 96 hours with 0, 1, 10, 20, 40, 80, or 160 \u0026micro;g/mL of SPNPs, HSPNPs, and HFSPNPs, 20 \u0026micro;L of MTT (5 mg/mL) was added to each well, and the mixture was incubated for an extra 4 h at 37\u0026deg;C. Following the formation of formazan crystals, 150 \u0026micro;L of DMSO was added, and a microplate reader was used to measure the absorbance at 570 nm. Wells devoid of any cells served as the blank, and the untreated cells served as the control. Five analyses were conducted on each sample at each concentration. The following formula was used to determine cell viability (%) =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{A}570（treated）-\\text{A}0}{\\text{A}570（untreated）-\\text{A}0}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003e570(treated)\u003c/sub\u003e is the absorbance of cells treated with samples, A\u003csub\u003e570(untreated)\u003c/sub\u003e is the absorbance value of the untreated cells, and A\u003csub\u003e0\u003c/sub\u003e is the absorbance value of the blank medium.\u003c/p\u003e \u003cp\u003eThe buffer capacity of the SPNPs, HSPNPs, and HFSPNPs was investigated via acid-base titration. Following the dispersion of SPNPs, HSPNPs, or HFSPNPs (0.2 mg/mL) in the aqueous NaCl solution (0.15 mol/mL), an aqueous NaOH solution (0.1 mol/mL) was added until the pH reached 10. Then, a solution of 0.1 mol/mL HCl (3\u0026ndash;5 \u0026micro;L) was added. The pH of the mixture was determined using a microprocessor pH meter following each addition of the HCl solution. The 0.2 mg/mL deionized water was regarded as the positive control and the 0.15 mol/mL NaCl solution as the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.9. In vitro drug release study\u003c/h2\u003e \u003cp\u003eUtilizing a dynamic dialysis procedure in PBS (pH 7.4) containing 0.5% Tween 80 and 10% ethanol, the siRNA\u003csup\u003eFAM\u003c/sup\u003e release capacity from SPNPs, HSPNPs, and HFSPNPs was investigated [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In a nutshell, 50 mL of the release media were dialyzed against 1 mL of free siRNA and siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, or siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs solution at 37\u0026deg;C in a shaking incubator at 120 rpm. The release medium was swapped out on a regular basis for equivalent amounts of brand-new release medium. Every experiment was conducted in triplicate, and the amount of released siRNA was determined using a fluorescence spectrometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Study on the pharmacodynamics characteristics of SPNPs, HSPNPs, and HFSPNPs\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.10.1. In vitro cellular uptake characteristics of nanoparticles\u003c/h2\u003e \u003cp\u003eIn order to confirm that the nanoparticles could target the liver, SPNPs, HSPNPs, and HFSPNPs were labeled with siRNA\u003csup\u003eFAM\u003c/sup\u003e, a simulated fluorescent marker. LO2 cells and SMMC-7721 cells were seeded on coverslips in 24-well plates for the qualitative cellular uptake study. The cells were then separated into the following groups: free siRNA\u003csup\u003eFAM\u003c/sup\u003e, siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs with the antagonist HA (or FA) (HA and FA pretreatment at a concentration of 1 mg/mL). The cells were exposed to new medium containing free siRNA\u003csup\u003eFAM\u003c/sup\u003e or siRNA\u003csup\u003eFAM\u003c/sup\u003e nanoparticles (siRNA\u003csup\u003eFAM\u003c/sup\u003e concentration, 100 nM) for 1, 2, or 4 h following 24 h of cell attachment. After discarding the media, the cells underwent three rounds of cold PBS washing, were fixed for 10 min using cold 4% paraformaldehyde, and their nuclei were then stained for 20 min with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) in order to identify them. The coverslips were then gently taken out of the wells and put onto the glass slides. A confocal laser scanning microscope was used to take the pictures. Six-well plates were used to seed LO2 cells for the quantitative cellular uptake investigation. Following a 24-hour incubation period, the cells were subjected to several siRNA\u003csup\u003eFAM\u003c/sup\u003e forms utilizing the identical procedure as the CLSM investigation. As a negative control group, cells that were not given medication were employed. Three cycles of cold PBS washing followed by trypsinization and 3 min at 1300 rpm centrifugation were performed on the cells. The cells were centrifuged after being cleaned with PBS. The cells were subsequently identified using flow cytometry after being resuspended in 500 \u0026micro;L of PBS solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.10.2. In vitro gene silencing\u003c/h2\u003e \u003cp\u003eSMMC-7721 cells were cultivated in 2 mL of DMEM with 10% FBS for 24 h after being plated at a density of 4\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well in 6-well plates. The cells were allowed to incubate for 4 h before being incubated for a further 20 h with fresh medium after the medium was replaced with opti-MEM containing siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs (1:1, 5:1, 10:1, and 20:1).\u003c/p\u003e \u003cp\u003eThe lysis buffer was used to extract the transfected cell proteins. Proteins were separated via gel electrophoresis after being put in equal amounts into the wells of a 10% SDS-PAGE gel. Upon transferring the proteins from the gel to the polyvinylidene difluoride membrane, the membranes were blocked for 60 min with 5% BSA. The membranes underwent 16 h of incubation at 4\u0026deg;C with BMP7 rabbit antibody (1:1000) and β-actin rabbit antibody (1:5000), followed by 60 min of secondary antibody treatment. Applying an enhanced ECL detection technique, the expression of BMP7 was identified. A Mini Chemi610 Imaging System was used to identify the western blot signals. Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e displayed the original western blots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.10.3. Protective studies of siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs in HCC tumor\u003c/h2\u003e \u003cp\u003eAfter being collected, SMMC-7721 cells were suspended in PBS at a density of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL. In order to initiate an in vivo focused experimental inquiry, female C57BL/6 mice were infected with 0.1 mL cell solution in the axilla until the tumor volume expanded to (100\u0026thinsp;\u0026plusmn;\u0026thinsp;20) mm\u003csup\u003e3\u003c/sup\u003e. 4 groups (n\u0026thinsp;=\u0026thinsp;5) of mice were randomly assigned: 1. control group (saline); 2. siBMP7/SPNPs; 3. siBMP7/HSPNPs; and 4. siBMP7/HASPNPs. Every 2 days, an intravenous injection of 200 \u0026micro;L of either the standard saline or the various siBMP7 formulations (equal to 0.33 mg/kg/day) was given into the tail four times in a row. Following the fourth dosage, the mice were put to sleep after an 8-hour fast (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The mice's primary organs and tumor tissues were completely removed, and the tumor tissues were weighed. For further H\u0026amp;E staining, all tissues were fixed in a 10% formalin solution. To identify the apoptosis of SMMC-7721 cells, cryostat sections of the tumor were cut into 10 \u0026micro;m thick sections and stained with Acridine orange (AO). These sections were then seen using a Leica DM2500 fluorescence microscope.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.10.4. Western blotting analysis\u003c/h2\u003e \u003cp\u003eFor western-blot analysis, RIPA buffer with 1 mM PMSF and 1% protease and phosphatase inhibitors were used to lyse the frozen tumor samples. The total protein concentration was assessed by the BCA method, and equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Incubation with the primary antibodies against BMP7 (1:1000) and β-actin (1:4000) was employed at 4\u0026deg;C overnight. The membranes were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. Densitometry using ImageJ software was calculated for quantification, and the densitometry results were normalized relative to the β-actin bands. The original western blots were shown in Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.10.5. In vitro safety evaluation\u003c/h2\u003e \u003cp\u003eFor in vitro safety evaluation, the tumor tissues and major organs of mice were dissected and fixed in 4% paraformaldehyde at room temperature for 24 h. Sections were prepared by paraffin embedding and H\u0026amp;E staining and observed and photographed under a CKX41 Optical microscope.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were performed at least in triplicate and expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical comparisons between groups were carried out using a one-way analysis of variance followed by the Student\u0026rsquo;s t-test. Values of \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Separation, purification and chemical compositions of SPS\u003c/h2\u003e \u003cp\u003eAfter 95% ethanol extraction, 5% alkali extraction, ethanol precipitation, and deproteinization, 37.7 g of crude polysaccharide SPS (yield 7.54% of 500 g of dry material) was produced from dried safflower. Following SPS fractionation using DEAE-cellulose ion exchange chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), two glycoconjugates, known as SPS-a and SPS-b, were obtained and eluted at distilled water and 0.25 M NaCl, respectively. A SephadexG-100 column was used to further purify SPS-a and SPS-b in order to produce a significant polysaccharide fraction known as SPS-1 (2.92 g, yielding 0.58% of 500 g dried material) and SPS-2 (1.72 g, yielding 0.34% of 500 g dried material)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). For the next physicochemical and animal tests, the peaks of SPS-1 and SPS-2 were collected, dialyzed, and dried. Purified polysaccharide SPS-1 and SPS-2 have respective carbohydrate contents of 94.2% and 92.1%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe absence of an absorption peak in the UV spectra of SPS-1 between 200 and 280 nm suggests that the polysaccharide lacked protein and nucleic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In the present study, we focused on the SPS-1 (subsequently replaced by SPS) fraction due to its easy access to enrich.\u003c/p\u003e \u003cp\u003eStandard curves of glucose and uronic acid are all shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. The composition of the monosaccharide can be determined by the retention time. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC、\u0026nbsp;Tab. and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the derivatives of monosaccharide standard D-glucose (Glu), D-galactose (Gal), D-xylose (Xyl), D-galacuronic acid (Gala), D-mannose (Man), L-rhamnose (Rha) and L-arabinose (Ara) showed absorption peaks at about 26.025, 29.398, 30.700, 22.450, 12.759, 17.051 and 32.137min, respectively. By comparing SPS-1 and SPS-2 polysaccharide maps, the monosaccharide composition can be determined by corresponding to them near the partially identical position, and the molar ratio of each monosaccharide can be calculated from the retained peak area. SPS-1 monosaccharide components respectively Man, Glu, Xyl, Ara, Gal, Rha, the mole ratio is: 5.36:8.72:4.61:1.96:19.36:4.52. The monosaccharide components of SPS-2 were Rha, Glu, Gal, Ara and Man with molar ratios of 3.00:7.12:5.88:1.16:4.02.\u003c/p\u003e \u003cp\u003eThe molecular weight of polysaccharide chromatogram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD(a)\u0026amp;(b) and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The retention time of SPS-1 was 20.269 min, the retention time of SPS-2 was 17.789 min, the MW of SPS-1 was 5546Da, Mn was 2984Da, and the polydispersis (D) was 1.925. Mw of SPS-2 is 30902Da, Mn is 13700Da, and D is 2.440. The results show that the larger the polydispersity, the wider the molecular weight distribution. This value is less than 1.05, where the sample is monodisperse, and greater than 1.05, where the sample is polydisperse. Therefore, both SPS-1 and SPS-2 are polydisperse samples, that is, the sample is widely distributed.\u003c/p\u003e \u003cp\u003eStudies had shown that if there is a three-dimensional helical structure in the polysaccharide molecule, it will form a complex with Congo red solution, resulting in the shift of the maximum absorption wavelength of Congo red solution. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, the maximum absorption wavelength of SPS-1 is close to that of Congo red phase, so there is no 3D helix structure, while the maximum absorption wavelength of SPS-2 shows a red shift, which is higher than that of Congo red, indicating that a complex can be formed. Thus, SPS-2 has a 3D helix structure.\u003c/p\u003e \u003cp\u003eThe results of infrared measurement of SPS-1 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF. There is a strong absorption peak at 3423cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating that there are intermolecular and internal hydrogen bonds of SPS-1. The absorption peak at 2924cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the special absorption peak of saccharide substances, 1425cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is caused by C-H angular vibration, 1051cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 950cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are C-O-C absorption peaks, 921.97cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the stretching vibration of the ring, and 895.45cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the C-H angular vibration of pyranose. It was judged to be the residue absorption peak of β-D-man. The infrared measurement results of SPS-2 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, 3423cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has a smooth absorption peak, SPS-2 has intermolecular and internal hydrogen bonds, 2920cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the peak of C-H tensile vibration, is the absorption peak of saccharide, 1660cm-\u003csup\u003e1\u003c/sup\u003e is the absorption peak of C\u0026thinsp;=\u0026thinsp;O in -COOH. 1433cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the C-O stretching vibration in -COOH, 1050cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has an absorption peak, indicating that SPS-2 has pyanose, 900\u0026thinsp;~\u0026thinsp;1225cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has an absorption peak of C-O-C and -OH, 917cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the characteristic absorption peak of α-D-gal. 841cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is a C-H stretching vibration, which indicates that SPS-2 has α-D-galactopyranose.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, the absorption signal peak of SPS-1 near δ4.79 ppm is the tritium proton signal in solvent D\u003csub\u003e2\u003c/sub\u003eO, and the proton peak signal at δH4.69 ppm indicates that it belongs to the sub-peak signal of β-D-glu residue matrix. The concentration area of the signal peak is less than 5.0 ppm, indicating that it is a β-configuration glycosidic bond, but there is an inverted peak. This is in agreement with the IR spectroscopic results. The δH1.18 ppm and 1.16 ppm resonance signals in the high-field region were inferred to be a small amount of rhamnose signal peak. SPS-2 is similar to SPS-1. The absorption signal peak near 4.79 ppm is the tritium proton signal in solvent D\u003csub\u003e2\u003c/sub\u003eO, and δH5.06 ppm belongs to the proton signal of Man, indicating that SPS-2 belongs to the alpha configuration glycosidic bond, which is consistent with the infrared spectrum results. The δH1.23 ppm and δH1.16 ppm in the high-field region may be the resonance methyl proton signal peak, which is a trace of rhamnose signal.\u003c/p\u003e \u003cp\u003eStandard curve of KIO\u003csub\u003e4\u003c/sub\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH.SPS-1 and SPS-2 can be completely reacted by KIO\u003csub\u003e4\u003c/sub\u003e within 2 days, and the consumption of KIO\u003csub\u003e4\u003c/sub\u003e and the production of formic acid are shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The amount of CH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced by SPS-1 was 13.2 \u0026micro;mol, indicating that for every 1\u0026rarr;6 bonded glycosyl, the amount of 1\u0026rarr;2 or 1\u0026rarr;4 bonded glycosyl was 27.3 \u0026micro;mol, and the remaining 1\u0026rarr;3 bonded glycosyl was 9.5 \u0026micro;mol. In SPS-1, the 1\u0026rarr;6 linkage or non-reducing terminal group accounted for 26.4%, other oxidable 1\u0026rarr;2 or 1\u0026rarr;4 linkage glycosyl accounted for 54.6%, and the non-oxidable 1\u0026rarr;3 residue accounted for 19%. These results suggested that SPS-1 was mainly composed of glucans with 1\u0026rarr;2 or 1\u0026rarr;4 linkage glycosyl as the main chain. The amount of CH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e produced by SPS-2 was 26.12 \u0026micro;mol, indicating that for every 1\u0026rarr;6 bonded glycosyl, the amount of 1\u0026rarr;2 or 1\u0026rarr;4 bonded sugar group was 4.11\u0026micro;mol, and the remaining 1\u0026rarr;3 bonded glycosyl was 19.77 \u0026micro;mol. In SPS-2, the 1\u0026rarr;6 glycoside-linked or non-reducing terminal group accounted for 52.24%, the 1\u0026rarr;2 or 1\u0026rarr;4 glycoside-linked group accounted for 8.22%, and the non-oxidable 1\u0026rarr;3 residue accounted for 39.54%, indicating that SPS-2 was mainly composed of 1\u0026rarr;6 glucans bonded to the main chain. Secondly, ara-gal with the glycosyl main chain partially bonded at the 1\u0026rarr;3 position is present. The specific connection mode of SPS-1 and SPS-2 needs to be determined after Smith degradation and methylation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular weight and polydispersion index of SPS-1 and SPS-2\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMw(Da)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMn(Da)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2984\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30902\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.440\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of SPS-1 monosaccharide composition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResponse factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeak area\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eContent(mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e155.140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10549.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e163.362\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e119.512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9808.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e137.762\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e169.428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21132.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e265.768\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29.786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e165.248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40087.860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e590.054\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eXyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e31.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e173.850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8488.045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e140.503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAra\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e182.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5880.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of SPS-2 monosaccharide composition\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRetention time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResponse factor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePeak area\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eContent(mg/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.805\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e155.140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15285.930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e168.530\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e119.512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11761.550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e128.413\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e169.428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18316.426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e209.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e29.585\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e165.248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16267.695\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e198.444\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAra\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e182.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7859.102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.077\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOxidation of KIO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdded amount\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConsumption of KIO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProduction of CH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026rarr;6 amount of glycosyl bonded\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026rarr;2/1\u0026rarr;4 amount of glycosyl bonded\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u0026rarr;3 amount of glycosyl bonded\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPS-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPS-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e19.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Synthesis and characterizations of the SPS-PEI conjugate\u003c/h2\u003e \u003cp\u003eThe hydrophobic moiety CDI was selected to modify the water-soluble SPS and produce the amphiphilic SPS-PEI compound. To sum up it briefly, PEI was first created to functionally introduce the carboxylic acid (-COOH), and as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, it was subsequently conjugated with SPS through an esterification reaction. The \u003csup\u003e1\u003c/sup\u003eH-NMR and FT-IR analyses verified the chemical structure. The \u003csup\u003e1\u003c/sup\u003eH-NMR spectra for SPS-PEI, as displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, revealed additional peaks at 2.52\u0026thinsp;~\u0026thinsp;2.74 ppm that were attributed to the protons of PEI in addition to the distinctive SPS peaks between 3.7 and 5.3 ppm, indicating the successful attachment of PEI to SPS. FT-IR was used to confirm the synthesis of SPS-PEI in more detail. The FT-IR spectra of SPS and SPS-PEI, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, both showed a distinctive absorption band about 3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which is due to the stretching vibrations of the hydroxyl group (-OH). Nevertheless, the intensity of this band is much lower than that of SPS, suggesting that some hydroxyl groups react. When comparing SPS with SPS-PEI, the former revealed a characteristic deformation peak at roughly 1570 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the latter revealed a new peak at about 1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, linked to the stretching vibrations of the newly created ester carbonyl group (C\u0026thinsp;=\u0026thinsp;O). Consequently, the synthesis of the SPS-PEI conjugate was accomplished. When the conjugate begins to self-assemble into nanoparticles by intra/intermolecular interaction, the threshold concentration can be used to estimate the critical aggregation concentration (CAC). Pyrene was used as the probe in the fluorescence probe technique, which was used to investigate the self-aggregation behavior of the SPS-PEI conjugate. The intensity ratio (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e339\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003e335\u003c/sub\u003e) of the pyrene excitation spectra as a function of the logarithm of the conjugate concentration of SPS and PEI is trended in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. The concentration of SPS-PEI at the intersection of the two lines, or CAC value, was 6.46 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e mg/mL. This suggests that the SPS-PEI conjugate has the potential to be used as a hydrophobic drug carrier because it can self-assemble into nanoparticles in an aqueous solution at very low concentrations and maintain the micellar structure before delivering the drug to the intended sites under extremely diluted conditions in the whole blood circulation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Synthesis and characterizations of the siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs\u003c/h2\u003e \u003cp\u003eTo put it succinctly, FA was first created to functionally introduce the carboxylic acid (-COOH), and as Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows, it was subsequently conjugated with HA by an esterification reaction. The distinctive peak of FA, which can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, emerged between 6.6 and 8.6 ppm, indicating that FA was successfully grafted onto HA. In the FT-IR of HA-FA, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB demonstrates that the ester carbonyl group (C\u0026thinsp;=\u0026thinsp;O) stretching vibration peak appeared at 1579 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Additionally, the absorption peaks of the amidogen (-NH\u003csub\u003e2\u003c/sub\u003e) at 1640 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and carboxylic acid (-COOH) at 3280 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e exceeded the corresponding peak in HA, indicating the synthesis of FA and CD44 receptors double targeting polymer HA-FA.\u003c/p\u003e \u003cp\u003eThrough electrostatic contact, the cationic SPNPs, HSPNPs, and HFSPNPs can form complexes with siRNA\u003csup\u003eFAM\u003c/sup\u003e. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, roughly spherical NPs can be generated by combining the siRNA\u003csup\u003eFAM\u003c/sup\u003e aqueous solution in the nuclease-free water with the SPNPs, HSPNPs, and HFSPNPs. The physiochemical characteristics of the complexes that formed were modified by adjusting the weight ratios of SPNPs, HSPNPs, and HFSPNPs to siRNA\u003csup\u003eFAM\u003c/sup\u003e. The resulting siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs complexes exhibit higher zeta potential as the weight ratios increase from 1:1 to 1:20, according to the DLS data in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. This occurs because more cationic SPNPs, HSPNPs, and HFSPNPs were utilized. The weight ratio of \u0026ge;\u0026thinsp;0.1 indicated a positive charge for the siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs. The proper particle size of the NPs is also important for efficient siRNA\u003csup\u003eFAM\u003c/sup\u003e delivery [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When the weight ratio decreases, the siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs develop into larger particles. The 100\u0026thinsp;~\u0026thinsp;200 nm diameter of the positive surface-charged siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs complexes is appropriate for cell uptake [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterizations of blank NPs and siRNA-loaded NPs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSize (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEE of siRNA (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e101.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.207\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/SPNPs (1:20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e132.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.245\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/SPNPs (1:10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e141.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.271\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;7.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/SPNPs (1:5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e148.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.304\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/SPNPs (1:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-7.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e160.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.251\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e67.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSPNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e110.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.263\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HSPNPs (1:20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e139.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.338\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84.1\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HSPNPs (1:10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e149.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.372\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HSPNPs (1:5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-7.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e157.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.398\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HSPNPs (1:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-11.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e172.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.034\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHFSPNPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e105.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.252\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HFSPNPs (1:20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e137.3\u0026thinsp;\u0026plusmn;\u0026thinsp;7.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.319\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HFSPNPs (1:10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e146.9\u0026thinsp;\u0026plusmn;\u0026thinsp;8.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.379\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HFSPNPs (1:5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e155.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.402\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esiRNA/HFSPNPs (1:1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e-10.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e176.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.351\u0026thinsp;\u0026plusmn;\u0026thinsp;0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Biocompatibility investigation\u003c/h2\u003e \u003cp\u003eWhen it came to the possible uses of nanocarriers in biomedicine, biocompatibility was taken into account. In this case, LO2 and SMMC-7721 cells' cell viabilities were assessed during a 48-hour incubation period with varying quantities of SPNPs, HSPNPs, and HFSPNPs. The outstanding safety of SPNPs, HSPNPs, and HFSPNPs is demonstrated by Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026amp;D, which shows that the cell viabilities of LO2 and SMMC-7721 cells were remained above 85% with concentrations of these substances ranging from 3.1 \u0026micro;g/mL to 100 \u0026micro;g/mL. In addition, since the nanocarriers come into correspondence with blood after being injected intravenously, serum stability is an essential indicator for nanocarriers. Utilizing serum relative turbidity, the stability of HFSPNPs, HSPNPs, and SPNPs was examined. Uncoated SPNPs interacted with serum more over time and had a reversely high positive charge, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA. On the other hand, following coating, there was a considerable decrease in the interaction between serum and HSPNPs and HFSPNPs, and this interaction did not change much as time passed. Time of the body for the circulation of nanoparticles was prolonged when their binding to non-specific proteins was decreased. Through acid-base titration, the buffer capacities of SPNPs, HSPNPs, and HFSPNPs were determined. The results demonstrate that all of the NPs have a significantly higher proton buffering capacity than NaCl, yet when compared to HFSPNPs, SPNPs and HSPNPs have a lower buffering capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Relatively speaking, HSPNPs and SPNPs have less amine groups than HFSPNPs, which could be one explanation. By using the \"proton sponge\" effect, HFSPNPs can boost the swelling of endocytic vesicles and transport siRNA into the cytoplasm, as demonstrated by this analysis of their strong proton buffering capacity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.5. In vitro drug release study\u003c/h2\u003e \u003cp\u003eThe in vitro drug release characteristics of free siRNA\u003csup\u003eFAM\u003c/sup\u003e, siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA. The findings showed that, after just 8 h, almost 87% of the free siRNA\u003csup\u003eFAM\u003c/sup\u003e was released. The release pattern of siRNA/HFSPNPs, on the other hand, was biphasic. A rapid release of 30% siRNA\u003csup\u003eFAM\u003c/sup\u003e from HFSPNPs happened in the first 8 h, while a slow release of 54.1% took place during the next 64 h. This is most likely due to the fact that the first burst release of siRNA\u003csup\u003eFAM\u003c/sup\u003e was caused by partially absorbed siRNA on the surface or siRNA\u003csup\u003eFAM\u003c/sup\u003e entrapped close to the surface of SPNPs, HSPNPs, and HFSPNPs, while the sustained release was caused by the amorphous form of siRNA that was imprisoned inside HFSPNPs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The findings suggested that HFSPNPs might find potentially relevance as a siRNA sustained release carrier.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Study on the pharmacodynamics characteristics of SPNPs, HSPNPs, and HFSPNPs\u003c/h2\u003e \u003cp\u003eThe cellular absorption of various siRNA\u003csup\u003eFAM\u003c/sup\u003e formulations in SMMC-7721 and LO2 cells was investigated in the present work using flow cytometry and confocal laser scanning microscopy (CLSM). The antagonist of choice was HA and FA owing to its strong affinity for the CD44 and FA receptors. Green fluorescence in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC\u0026amp;D suggested that LO2 and SMMC-7721 cells could receive siRNA\u003csup\u003eFAM\u003c/sup\u003e-loaded NPs. The time-dependent cellular absorption of siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, suggested that the NPs accumulated inside cells gradually to exert their therapeutic effects. Similar results were also observed in SMMC-7721 cells. More importantly, the higher accumulation efficiency of siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs compared with siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs might be ascribed to the high affinity of the HFSPNPs for CD44 and FA. Furthermore, LO2 and SMMC-7721 cells presented a greater decrease in green fluorescence intensity with the prior addition of HA and FA. This was because HA (or FA) could competitively bind to CD44 (or FA) and restrain the endocytosis of CD44 (or FA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs predicted, after 4 h of incubation with siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs, and siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs, the green fluorescence intensities in SMMC-7721 cells were stronger than those in LO2 cells, which somewhat supports CD44 (or FA) -mediated endocytosis. Quantitative flow cytometry tests were carried out to further validate the liver-targeting capacity of SPNPs, HSPNPs, and HFSPNPs. The uptake of each siRNA\u003csup\u003eFAM\u003c/sup\u003e formulation in SMMC-7721 cells was time-dependent, as predicted, and at each time point, the mean fluorescence intensity of the siRNA\u003csup\u003eFAM\u003c/sup\u003e/HFSPNPs group was significantly higher than that of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). These outcomes agreed with the CLSM findings.\u003c/p\u003e \u003cp\u003eAfter confirming the high siRNA uptake utilizing NPs, we employed siBMP7 to assess the effectiveness of gene silencing in SMMC-7721 cells via western blot analysis. The silencing efficacy varies based on the weight ratios, as Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF\u0026amp;G illustrating. The CLSM and flow cytometry showed that as the weight ratios of siBMP7 to HFSPNPs growing, more siBMP7 was internalized into the cells, resulting in a drop in BMP7 levels in the cells. In comparison to the control group, the siBMP7/HFSPNPs with a weight ratio of 20:1 could lower the BMP7 expression to about 20%. Based on the in vitro experiment's poor absorption capacity, we eliminated the group with free siRNA\u003csup\u003eFAM\u003c/sup\u003e from the in vivo trial.\u003c/p\u003e \u003cp\u003eThe efficacy of enhancing site-specific delivery of siRNA and HCC prevention was compared among control, siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs groups. In the in vivo model, SMMC-7721 cells were sequentially pretreated for 7 days in all animals except those in the control group. After that, the model was constructed with three distinct formulations and saline as a control. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u0026amp;B showed the differences in body weights and tumor volumes. Each carried the same body weight for the duration that the tumor was borne. All body weights, with the exception of the control group, began to develop less quickly after the 7th day of formulation administration. Tumor volume remained same on day 7 but altered on day 9, while siBMP7/HFSPNPs showed total tumor inhibition before the experiment ended, indicating that treatment with siBMP7/HFSPNPs might more successfully inhibit tumor growth. Ultimately, the siBMP7/HFSPNPs inhibition rate may reach 69.5% based on the tumor quality displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC\u0026amp;D. To bolster the protective impact of siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs, histological studies were conducted. As demonstrated by H\u0026amp;E staining of tumor sections, different formulations reduced severe lesions, with the siBMP7/HFSPNPs group showing the largest preventative advantages (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAs seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eB, more cells were participating in the autophagic process when treated with siBMP7/HFSPNPs as compared to cells treated with siBMP7/SPNPs and siBMP7/HSPNPs. These cells also displayed greater apoptosis and stronger red fluorescence spots. The results imply that the HA-FA coating can promote carrier uptake by SMMC-7721 cells and thus trigger the demise of cancerous cells. Additionally, from a pharmacological perspective, western blot might show that HFSPNPs can mute the BMP7 gene more successfully, enabling increased siBMP7 distribution to cells through Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC\u0026amp;D.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Safety of nanoparticles in vivo\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e, several NPs formulations demonstrate remarkably in vivo safety. Tumor-bearing mice treated with siBMP7/SPNPs, siBMP7/HSPNPs, and siBMP7/HFSPNPs showed no significant injury to the kidney, liver, spleen, lung, or heart aside from the tumor tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNatural polysaccharides are superior to other non-viral vectors for gene delivery for the reason they are simple to prepare and produce, exhibit biological activity such as the ability to identify liver cells and macrophages, and are easily hydrolyzed and enzymolized in cells. The breakdown products of monosaccharides and amino compounds also have low toxicity and may even provide nutrients and promote cell growth. Polysaccharide recognition by cell surface receptors could speed up cell internalization after several naturally occurring polysaccharides in the amination reaction were designed and synthesized using the gene carrier with high transfection efficiency. This represents the establishment of a cationic polysaccharide carrier with low cytotoxicity and immunogenicity, good biodegradability, and high solubility in water, compared to those synthesized using other cationic polymers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Importantly, whereas certain polysaccharides have helical structures, they can hydrogen link with polynucleotide polymers to generate a hybrid three-strand helical polymer that can effectively prevent polynucleotide degradation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Naturally occurring polysaccharides have low toxicity and good biocompatibility, while PEI, a traditional transfection reagent, has a high transfection rate and significant cytotoxicity. Due to this, scientists combined the benefits of the two materials by modifying natural polysaccharides using PEI as the gene carrier [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince HA is an acidic mucopolysaccharide [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], it can be chemically or physically changed to create derivatives that have a variety of uses as materials for drug delivery vehicles. HA and its derivatives have found widespread use as drug carrier materials because of their exceptional biocompatibility, biodegradability, receptor binding, non-toxicity, and immunogenicity. Human cells have a high number of HA receptors. HA and its derivatives can target specific cell surface receptors to deliver medicines to those cells. Many receptors are under investigation, such as the HA receptor-1 on lymphatic endothelial cells, the CD44 receptor, the HA-mediated motivation (RHAMM) receptor, and the HA endocytosis receptor [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Many tumor cells have overexpressed CD44 and RHAMM receptors on their surface, which when paired with HA-modified vectors allows for active drug vector targeting. An amphiphilic HA-deoxy acid linking self-assembly of redox-sensitive micelles, for instance, was created by Li et al. to deliver paclitaxel specifically into cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The glycoprotein known as the FA receptor is expressed at low to moderate levels in the lungs, kidneys, and choroids but is virtually absent from other normal tissues. In the majority of malignant tumors, including ovarian, cervical, kidney, breast, and colon cancers, the high-affinity FA receptor was significantly expressed. As a naturally occurring ligand of the FA receptor, FA is currently inexpensive, non-toxic, and simple to access and alter. In addition, it displays excellent biocompatibility, robust FA receptor binding, and minimal immunogenicity. Additionally, the FA molecule is stable and may sustain a long blood circulation or storage period while maintaining a reasonably high receptor affinity. Direct coupling of FA to drug molecules and coupling of FA on the surface of the drug carrier are the two main methods used by the FA receptor-mediated active targeted drug delivery system to target tumor locations. The solubility of commonly used anti-tumor drugs is normally lower in water under physiological settings. This is due to the direct coupling of FA to the drug molecules, which further reduces the solubility of the medication in water and ultimately reduces its bioavailability. Therefore, the coupling of FA to the surface of a drug carrier strategy has research value; currently, the FA receptor is the main focus in research on the active targeting drug delivery system.\u003c/p\u003e \u003cp\u003eTo sum up, the research successfully created self-assembled nanoparticles. HFSPNPs could effectively accumulate in hepatic cells by HA-FA-mediated uptake, according to studies done both in vitro and in vivo. This suggests that HFSPNPs could be a viable delivery mechanism for drugs to be delivered to the liver, where they might exhibit their protective effects. In SMMC-7721 cells, the siBMP-7/HFSPNPS particles showed strong BMP7 knockdown along with strong in vitro and in vivo stability, strong gene loading capacity, high cell uptake, transfection efficiency, and tumor-targeting properties. Even though no harm was done to other organs, the tissue distribution staining experiment demonstrated that HFSPNPs may increase the amount of siBMP7 that accumulates in the tumor. The release of siBMP7 from the nanoparticles allowed for the achievement of the gene therapy goal by promoting apoptosis in cancer cells and reducing the expression of BMP7 protein in tumor tissue cells. These findings suggest that HFSPNPs may be used as an HCC tumor-targeting drug in the treatment of HCC, which has implications for more research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.cn) for their assistance with English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by\u0026nbsp;the Laboratory Animal Center, Heilongjiang University of Chinese Medicine and also followed the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe figures and tables supporting the results of this study are included in the article, and the original datasets are available from the first author or corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China under Grant number 81603418; and the Central Government Supports Local College Reform Projects under Grant number 2020YQ05.\u003c/p\u003e\n\u003cp\u003eThe funding agencies had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaotian Bai: Writing the original manuscript draft; Conceptualization; Data curation; Visualization.\u003c/p\u003e\n\u003cp\u003eJing Yang: Supervision; Resources; Funding acquisition.\u003c/p\u003e\n\u003cp\u003eJunhao Zhang: Data validation;\u0026nbsp;Software; Data curation\u003c/p\u003e\n\u003cp\u003eRui Wang: Formal analysis; Methodology; Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaotian Bai (ORCiD ID: 0000-0002-7158-7668): College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email: [email protected]\u003c/p\u003e\n\u003cp\u003eJing Yang (ORCiD ID: 0000-0002-9705-3883): College of Basic Medical Science, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email:[email protected]\u003c/p\u003e\n\u003cp\u003eJunhao Zhang: College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China. Email: [email protected]\u003c/p\u003e\n\u003cp\u003eRui Wang (ORCiD ID: 0000-0003-4770-3515): College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China; College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang 150040, China; Key Laboratory of Basic and Application Research of Beiyao, Heilongjiang University of Chinese Medicine, Ministry of Education, Harbin, Heilongjiang 150040, China. Email: [email protected]\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL.A. Torre, F. Bray, R.L. 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Biomaterials. 33 (7) (2012) 2310\u0026ndash;2320.\u003c/li\u003e\n\u003c/ol\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":"Dual-target, Safflower polysaccharide, Hepatocellular carcinoma, Self assembled nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-6119667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6119667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHepatocellular carcinoma (HCC) is the second most common cause of cancer-related death with chemotherapy and traditional surgery showing limited effectiveness. The present work aimed to study the feasibility of safflower polysaccharide (SPS) as an instinctive liver-targeting drug delivery carrier with applications in HCC. SPS-polyethyleneimine (SPS-PEI), hyaluronic acid-SPS-polyethyleneimine (HA-SPS-PEI), and hyaluronic acid-folic acid-SPS-polyethyleneimine (HA-FA-SPS-PEI) conjugates were synthesized by an esterification reaction and characterized by conventional methods. SPS-PEI, HA-SPS-PEI, and HA-FA-SPS-PEI self-assembled nanoparticles (SPNPs, HSPNPs, and HFSPNPs, respectively) and siRNA-loaded SPNPs, HSPNPs, and HASPNPs (siRNA\u003csup\u003eFAM\u003c/sup\u003e/SPNPs, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HSPNPs and siRNA/HASPNPs, respectively) were fabricated with a roughly spherical shape, with sizes were ranging 100\u0026thinsp;~\u0026thinsp;200 nm in aqueous solution. Compared with free siRNA\u003csup\u003eFAM\u003c/sup\u003e, siRNA\u003csup\u003eFAM\u003c/sup\u003e/HASPNPs displayed enhanced serum stability, hypo toxicity, and a sustained release of siRNA\u003csup\u003eFAM\u003c/sup\u003e over 64 h. In the in vivo cellular uptake behavior study, the HASPNPs showed excellent HCC tumor-targeting capability because of the specific recognition by the folic acid and hyaluronan receptors (CD44) overexpressed on the HCC tumor membrane. The tissue staining of siRNA\u003csup\u003eFAM\u003c/sup\u003e/HASPNPs in mice further demonstrated that HASPNPs could distinctly enhance the distribution of siRNA\u003csup\u003eFAM\u003c/sup\u003e into the HCC tumor. Our results indicate that HASPNPs may serve as a promising HCC tumor-targeting drug delivery carrier for HCC prevention.\u003c/p\u003e","manuscriptTitle":"Synthesis, characterization, and application of self-assembled safflower polysaccharide nanoparticles as liver targeting drug delivery carrier","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-17 09:39:21","doi":"10.21203/rs.3.rs-6119667/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":"c85f31f1-e670-495d-8c59-ff945ac688ab","owner":[],"postedDate":"March 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45771836,"name":"Biological sciences/Cancer"},{"id":45771837,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-04-30T08:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-17 09:39:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6119667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6119667","identity":"rs-6119667","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}