1. Introduction
Nanodelivery systems are a promising in the biomedical field. Compared to traditional drug carriers, nanocarriers improve the pharmacokinetics and biodistribution of therapeutic drugs, thereby maximizing their concentration at the target and minimizing toxicity [
1]. Specifically, nanoparticles effectively improve the solubility and bioavailability of drugs, prolong the residence time of drugs in the body, and reduce their side effects. Common inorganic nanocarriers include mesoporous silica nanoparticles (MSNs), gold nanoparticles, carbon nanoparticles, magnetic nanoparticles, and quantum dots [
2]. Among these nanocarriers, MSNs have received particular attention from many researchers because they can address some of the inherent drawbacks of therapeutics, including limited bioavailability, short cycle life, and adverse biodistribution [
3]. In the field of drug delivery, MSNs have many advantages: (1) as micro-reservoirs, they have good ability to contain guest molecules and can release the cargo carried under physiological conditions; (2) they have adjustable pore sizes, so they can be loaded with different carriers; (3) they can achieve targeted delivery and controlled release through surface modification; (4) the combination of loading magnetic and fluorescent compounds can realize the dual functions of drug delivery and biological imaging at the same time [
4,
5,
6,
7]. MSN is a type of nanomaterial that is easy to modify and can be combined with other materials to form nanocarriers with multiple functions, such as surface modification. Biomedical applications of MSNs include improving drug solubility, as vectors for controlled/targeted transport, as carriers for diagnostics, and as antigen carriers and adjuvants for vaccine delivery.
The use of poly-dopamine (PDA) to modify material surface properties offers new possibilities for designing nano-carriers [
8]. Using PDA to modify the surface properties of nanoparticles is more advantageous than using other surface functional materials. The synthesis of PDA requires simple and gentle, due to its unique adhesion properties, no organic solvents are required, and the synthesis of PDA core-shell nanoparticles can be completed by stirring dopamine hydrochloride in Tris-HCl buffer solution at pH 8.5 [
9]. In addition, a variety of different PDA surface modifications can be designed by adjusting basic parameters such as pH, temperature, dopamine concentration, oxidant, and reaction time [
10]. Second, the drug loading capacity of nanoparticles can be significantly improved by modification of PDAs, which have rich catechol/quinone structures that bind functional molecules to nanoparticles through physical bonding (π-π or hydrogen bonding) or chemical bonding (Michael addition or Schiff base reaction) [
11]. In addition, secondary modifications such as polyethylene glycolation can be achieved with the help of PDAs, as it can react with thiols or amino-containing compounds using Michael addition or a Schiff base [
12]. The uniqueness of PDA modifications provides nanoparticles with better hydrophilicity, biocompatibility, and biodegradability, as well as photothermal conversion and reactive oxygen species (ROS) scavenging. This allows PDA-modified nanoparticles to have a variety of functions, from targeting, imaging, and chemical processing (CT) to photodynamic therapy (PDT), photothermal therapy (PTT), tissue regenerative capacity, anti-inflammatory, and antioxidant effects, all of which can play a role in these areas. Therefore, PDA-modified nanoparticles are widely used in cancer treatment, antibacterial applications, therapeutic diagnosis, tissue repair, and other aspects [
13,
14,
15,
16].
There are different types of cells in the liver, namely, hepatocytes, Kupfer cells, hepatic stellate cells, and sinusoidal endothelial cells, among others. There are different types of receptors on the surface of these cells, and by binding the drug to a targeted portion that matches the receptor on the surface of hepatocytes, it is possible to precisely target the drug to the liver. Liver-targeted drug delivery systems (HTDDS) can be achieved using a variety of nanocarriers. HTDDS can not only distribute drugs to the liver but also improve the bioavailability of drugs and reduce adverse reactions to drugs [
17]. ASGPR receptors are mainly expressed by hepatocytes and are less distributed in extrahepatic cells [
18]. It is one of the most studied targets for selective delivery of anti-cancer drugs to hepatocellular carcinoma (HCC) [
19]. ASGPR binds to galactose or galactosamine with high affinity and can also be combined with carbohydrates including glucose and polysaccharides and polymers with sugar residues. Thus, galactose, lactose, lactoferrin moiety, and a variety of carbohydrates with repeating galactose or glucose units are used as liver-targeted drug delivery systems [
20].
Aspirin (Asp) is a nonsteroidal anti-inflammatory drug that can reduce prostaglandin biosynthesis by inhibiting the activity of COX1 and COX2 (cyclooxygenase1 and cyclooxygenase2). Compared to other NSAIDs, low-dose aspirin (75–100 mg) exerts an antiplatelet drug effect by covalently modifying COX1 expressed in mature platelets. High doses of aspirin (650–1300 mg) can cause COX2 acetylation in inflammatory cells, exerting analgesic and anti-inflammatory effects [
21]. There is experimental and clinical evidence that aspirin also has the characteristics of chemoprevention and chemotherapy for cancer. Epidemiological studies in both the general population and high-risk groups have shown a correlation between regular aspirin use and a decrease in the incidence of HCC. Furthermore, regular aspirin use among HCC patients has been found to effectively reduce both recurrence and mortality rates [
22]. In addition, preliminary preclinical studies have shown that aspirin’s mechanism of action on HCC may be related to its antiplatelet and anti-inflammatory activities [
23,
24].
Based on this, MSNs were prepared by the “one-pot two-phase layering method”, functionalized and modified, and the “gated switch” PDA was coated on the surface of MSNs. Then the secondary reactivity of polydopamine was used to connect the target ligand Gal to the surface of nanoparticles, details are shown in
Scheme 1. Aspirin was selected as a model drug to construct a nano-drug with pH sensitivity and targeting, as shown in
Scheme 2. The relevant characteristics, biological safety, drug release mechanism, and pharmacodynamic effect on liver cancer cells were preliminarily studied, and analyzed from quality evaluation to pharmacodynamic evaluation, from in vitro to in vivo, to provide a reference for the research of nanomedicine based on secondary modification of polydopamine.
3. Materials and Methods
3.1. Chemicals
The chemical reagents required for this experiment are provided by the supplier and used according to the usage specifications. Dopamine hydrochloride, triethanolamine (TEA), Tris buffer (pH = 8.5), D-(+)- galactosamine hydrochloride, and ether were all purchased from McLean Technology Co., Ltd. (Shanghai, China). Anhydrous ethanol, methanol, and cyclohexane were all purchased from Tianjin Damao Reagent Factory (Tianjin, China). Tetraethyl orthosilicate (TEOS) and cetyltrimethyl ammonium chloride (CTAC) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Unless otherwise specified, all chemical reagents are of analytical grade and used without other treatment.
3.2. Animals
Twenty-four 5-week-old KM mice with no specific pathogen level were purchased from Slack Jingda Experimental Animal Co., Ltd. (Changsha, China). (License number: SYXK (Guangdong) 2022-0125), and the quality certificate number of experimental animals was NO.110727201003661. The experimental animals are kept in the pathogen-free laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University. This experiment was approved by the Experimental Animal Ethics Committee of Guangdong Pharmaceutical University and strictly followed the requirements of the Guidelines for Ethical Review of Experimental Animal Welfare (GB/T35892-2018) to fully protect the welfare of experimental animals.
3.3. Synthesis of MSN, PDA-MSN, and Gal-PDA-MSN
A mixture of 36 mL of distilled water, 24 mL (25 wt%) of CTAC solution, and 0.18 g of TEA was stirred at 70 °C for 1 h at a stirring rate of 150 rpm/min. Subsequently, 20 mL of TEOS and 2 mL of cyclohexane were added to react. The reaction temperature was set to 70 °C with a magnetic stirring rate of 150 rpm/min, and the reaction time was 24 h. Following the reaction, a high-speed centrifuge was used to spin at 13,000 rpm/min for 20 min to obtain a white precipitate. The crude product was washed three times with ethanol to eliminate any residual reactants. The collected products were washed twice with acetone at an oil bath temperature of 50 °C and continuously stirred for 12 h to remove the template CTAC. Subsequently, the products were washed twice with ethanol to eliminate any residual acetone. Finally, the resulting product was freeze-dried to obtain a powdered MSN product.
MSN (50 mg) was dispersed in 50 mL Tris-HCl buffer (pH 8.5, 10 mmol/L) using ultrasound. Then, dopamine hydrochloride (25 mg) was added and stirred for 20 min. Subsequently, 2 mL of H2O2 (30 wt%) was added, and stirring was continued for 3 h in the dark. After the reaction, the solid product of PDA-MSNs coated with PDA was centrifuged (12,000 rpm, 10 min) and washed with water to remove unpolymerized dopamine. PDA-MSNs are stored in the refrigerator at 4 °C for later use.
The PDA-MSNs (20 mg) were dispersed in 20 mL Tris-HCl buffer (pH 8.5, 10 mmol/L) using ultrasound. Galactosamine hydrochloride (40 mg) was then added, and the reaction was stirred for 0.5 h at room temperature. After the reaction, unreacted galactosamine hydrochloride was removed by ultrafiltration centrifugation, resulting in the preparation of Gal-PDA-MSNs.
3.4. Loading of Aspirin (Asp)
Dissolve 10 mg of aspirin in 5 mL of PBS buffer, then add 5 mg of MSN and magnetically stir for 12 h. MSN loaded with aspirin was centrifuged and freeze-dried for storage. The absorbance of the centrifuged supernatant at 222 nm was measured using an Ultraviolet spectrophotometer (Max M4, Meigu Molecular Instruments Co., Ltd., Shanghai, China). The content of Asp in the supernatant was calculated using the standard curve method. The loading number of aspirin in MSN@Asp was determined by the difference in aspirin content before and after.
Replace MSN with MSN@Asp, and prepare PDA-MSN@Asp and Gal-PDA-MSN@Asp according to
Section 2.3.
3.5. Drug Release Experiment of Drug-Loaded Nanoparticles In Vitro
The three nano-drugs were dispersed in 5 mL PBS buffer solution, then the mixture was transferred to 3500 Da dialysis bags, and then placed in 30 mL PBS buffer solution containing 1% SDS (pH = 7.4, 5.2), and slowly oscillated in a constant temperature oscillator at 37 °C and 100 rpm. At specific time points (1 h, 2 h, 3 h, 4 h, 12 h, 24 h, 48 h, and 72 h), 2 mL of precipitated solution was taken out from the outside of the dialysis bag, and then PBS buffer with the same volume was added into the dialysis bag. After the solution was diluted, the concentration of Asp was determined by ultraviolet-visible spectrophotometer at 222 nm, and the content of Asp in each group was calculated according to the standard curve equation, and the cumulative drug release curve was drawn for comparison. Three groups of parallel experiments were set up for each sample, and the results were averaged. The cumulative release rate is calculated by Formula (3):
Q: cumulative release rate of aspirin (%); V: sampling volume at a time (mL); Ci: the concentration of aspirin at the first sampling (mg/mL); V0: Total volume of buffer in dialysis bag (mL); Cn: the concentration of aspirin at the nth sampling (mg/mL); M: total dosage of drug-carrying system (mg).
3.6. Characterization of Nanoparticles
The size distribution and zeta potential of the prepared nanoparticles were measured by a laser particle size analyzer (Delsa, Beckman Technology Co., Ltd., Durham, NC, USA).
The morphology of the prepared nanoparticles was observed by scanning electron microscope (XFlash 6130, Carl Zeiss, Oberkochen, Germany) and transmission electron microscope (Tecnai G2 F20, Thermo Fisher Scientific, Waltham, MA, USA). A small number of MSNs, MSN-PDA, and Gal-PDA-MSNs powders were uniformly sprayed on the sample base affixed with double-sided conductive adhesive. The sample base was placed in the SEM instrument, and the optimal magnification and observation position were used to take surface morphology shots. A small amount of MSNs, MSN-PDA, and Gal-PDA-MSNs powders were dispersed uniformly in double-distilled water to obtain a sample suspension of moderate concentration, and a drop of about 8 μL of the sample suspension was suspended on the copper grid of the electron microscope, and after the solvent evaporated and dried up, the sample suspension was placed under the TEM to photograph its morphology using the optimal observation magnification and position.
The micropore structure and pore size distribution of the prepared nanoparticles were determined by nitrogen adsorption experiment (asap2460, American Mack Instruments Co., Ltd., Colonial Heights, VA, USA).
3.7. Preliminary Safety Evaluation
The human hepatoblastoma cell line (HepG2) was obtained from the cell bank of the China Academy of Sciences and cryopreserved in liquid nitrogen. The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL of streptomycin at 37 °C in a 5% carbon dioxide incubator.
After the in vitro cultured HepG2 cells covered the bottom 80% of the culture dish, the cells were inoculated in 96-well plates according to 8000–10,000 cells per well, and the cell status was observed using a microscope. After approximately 12 h of culture, 5, 10, 25, 50, 100, 200, and 400 μg/mL of MSN, PDA-MSN, and Gal-PDA-MSN nanomaterials were administered. Three replicate wells were set up for different concentrations of each nanomaterial. A blank group (medium without cells and drug to be tested, CCK8) and a positive control group (medium containing cells, CCK8, no drug to be tested) were also set up. The incubation was continued for 24 h after the administration of nanomedicine, then the supernatant was removed and a solution of CCK8 at a concentration of 10% configured with DMEM medium was added to each well and placed in an incubator to continue incubation for 1 h before terminating the incubation. The OD value of each well was measured at 450 nm on an enzyme marker and the survival rate was calculated according to the following formula.
As: absorbance of the experimental hole (culture medium containing cells, CCK-8, sample to be tested); Ac: absorbance of control well (medium containing cells, CCK-8, no sample to be tested); Ab: absorbance of the blank hole (culture medium without cells and samples to be tested, CCK-8).
One mL of fresh rabbit blood was taken and centrifuged at 3000 rpm/min for 10 min to collect the erythrocytes. The erythrocytes were washed with saline until the supernatant was completely colorless, and then the erythrocytes were subsequently reconstituted into 2% erythrocyte suspension with saline. The absorbance was measured by UV spectrophotometry using saline as the negative control and deionized water as a positive control. The nanoparticles were diluted into samples with concentrations of 50, 500, 1000, and 1500 μg/mL with saline, and 2 mL of the sample solution was mixed with 2 mL of 2% erythrocyte solution. After mixing, it was immediately placed in a thermostat for incubation at 37 °C ± 0.5 °C. The absorbance was measured at 540 nm by centrifugation of the supernatant solution after 4 h.
After a week of adaptive feeding, KM mice were randomly divided into four groups, with six mice in each group (half male and half female). Then, the control group was injected with 100 μL of normal saline through the tail vein, and the experimental group was injected with 100 μL of MSN, PDA-MSN, and Gal-PDA-MSN dispersion (dosage: 600 mg/kg), respectively. Then, the body weight was recorded, and the spirits and activities of the mice were observed every day. After feeding for 7 days under normal conditions, the mice were fasted for 12 h, anesthetized with ether, and blood was taken from the orbit. The mice were dissected and the main organs, such as the heart, liver, spleen, lung, and kidney, were taken out and weighed to calculate the organ index. After sampling, the tissue was stored at −80 °C for subsequent detection and analysis.
3.8. In Vitro Cellular Uptake
Fluorescent probe-labeled nanoparticles (C6-NPs) were prepared by replacing Asp with an equal amount of coumarin-6 (C6) under light-avoidance conditions according to
Section 2.4. HepG2 cells were seeded in 6-well plates at 1 × 10
6 cells/well and incubated for more than 12 h until 80% cell-adherent coverage was achieved, the original medium was aspirated and the wells were washed with PBS. The C6 solution and C6-NPs were diluted with a serum-free medium so that the fluorescein concentration was 1 μg/mL for both. After the cells in each group were given C6 and C6-NPs, they were incubated at a temperature of 37 °C for 4 h. At the end of the incubation, the fluorescein-containing medium was removed, and the cells were washed with PBS 2 times. Cells were digested with trypsin for 4 min and blown into cell suspension, centrifuged at 1000 rpm/min for 3 min, and then collected and washed twice with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min and collected by centrifugation. Finally, the cells were resuspended with PBS, and 500 μL was placed in a flow tube, and the uptake of coumarin by HepG2 cells was detected using flow cytometry.
3.9. Anticancer Effect In Vitro
HepG2 human hepatocarcinoma cells were selected as the experimental object, and commercial aspirin (Asp) was used as the positive control. The inhibitory abilities of Asp, MSN@Asp, PDA-MSN@Asp, and Gal-PDA-MSN@Asp were compared. 10,000 cells per well were seeded in a 96-well plate, and the cell state was observed by a microscope. After about 12 h of culture, four groups of Asp, MSN@Asp, PDA-MSN@Asp, and Gal-PDA-MSN@Asp were set up according to the experimental needs, with 5 wells in each group. Aspirin with the final concentration of 0.1, 1, 5, 10, and 20 mM was given in the form of liquid exchange. Through equivalent conversion, the content of Asp in free drug and nano-drug is ensured to be the same. After the drug was added, the culture was continued for 24 h, and the 10% CCK8 solution prepared with DMEM medium was added by changing the liquid, placed in an incubator, and continued to incubate for 1 h, then the culture was terminated. Measure the OD value of each orifice at 450 nm on an enzyme-labelled instrument, and calculate the cell inhibition rate according to the following formula:
As: absorbance of the experimental hole (culture medium containing cells, CCK-8, a drug to be tested); Ac: absorbance of control well (medium containing cells, CCK-8, no drug to be tested); Ab: absorbance of the blank hole (culture medium without cells and drugs to be tested, CCK-8).
Cell migration experiment. Three horizontal lines with equal spacing (about 0.5 cm) were horizontally drawn on the bottom of the 6-well plate with a marker. Cells in the logarithmic growth stage were digested into single-cell suspension by trypsin, diluted to 1 × 106 cells/well, then inoculated into 6-well plates respectively, and 2 mL of DMEM medium was added to each well, and finally put into an incubator with 37 °C and 5% CO2 for culture. The negative control group and four experimental groups (Asp, MSN@Asp, PDA-MSN@Asp, Gal-PDA-MSN@Asp) were set up, and each group was set up with three multiple holes. When the cell fusion degree was close to 100%, three vertical lines with equal spacing were drawn on the bottom of the 6-well plate with a sterile gun head of 100 μL, which are perpendicular to the horizontal lines drawn by the marker. The culture medium was removed, cells were washed with PBS buffer twice to remove the scratched cells, cell spacing was observed under the microscope, and photos were taken. Serum-free basic medium was added, the above was repeated after 48 h, and the scratch spacing was measured. Scratch healing rate = (scratch spacing before healing-scratch spacing after healing)/scratch spacing before healing × 100%.
3.10. Statistical Analysis
In this experiment, the data were analyzed and plotted by GraphPad Prism 9 software, the two groups were compared by t-test, and more than two groups were analyzed by One-way ANOVA. When p < 0.05, the difference was significant, and the data results were expressed by mean ± SEM.
4. Conclusions
In this study, PDA-modified and Gal-modified mesoporous silicon nanoparticles were constructed using inorganic nanomaterials, and Asp was loaded on the nanoparticles to be used for targeted therapy of liver cancer. The results show that the drug-loaded nanoparticles have a liver-targeting effect and pH-response-release effect, which can accurately act on liver cancer cells and avoid any effects on normal tissues. We preliminarily studied the related properties, biological safety, drug release mechanism, and anti-tumor efficacy (in vivo and in vivo) of Gal-PDA-MSN@Asp, and analyzed it from several aspects including quality evaluation and anti-tumor efficacy, both in vitro to in vivo, to provide references for human body and in vivo evaluation. Gal modification enables nanoparticles to target the salivary acid soup protein receptor on HepG2 to achieve a liver-targeting effect, while PDA modified on the MSN surface has the effect of pH response release, which can release Asp in the acidic microenvironment of a tumor. Compared with aspirin administration alone, Gal-PDA-MSN@AspPDA has a better inhibitory effect on liver cancer cells. In this study, the MSN surface modification strategy is simple, universal, and results in nano-drugs with new surface properties; these can be used as potential carrier platforms for targeted nano-drugs, providing a valuable reference for future targeted drug delivery systems developed according to the characteristics of the tumor microenvironment.