Abstract
To address the undesirable reactions associated with matrine (MAT) injection in clinical settings, a high-loading drug delivery system (DDS) based on pH-sensitive molecularly imprinted polymer (MAT@MIPs) was prepared for the first time. The imprinted materials containing recognition sites for the matrine were formed by using carboxyl-functionalized multiwalled carbon nanotubes as a supportive matrix and dopamine as a cross-linker due to its exceptional biocompatibility. Subsequently, the optimal reaction conditions and adsorption performance of MAT@MIPs were systematically investigated. The obtained polymers were characterized and evaluated by Fourier transform infrared spectrometry, scanning electron microscopy, elemental analysis, and thermogravimetric analysis. Results indicated that the MIPs demonstrated a favorable imprinting factor (2.36) and a high binding capacity (21.48 mg·g−1) for matrine. In vitro studies, we performed cell counting kit-8 assays in HepG2 cells, then the drug delivery capabilities of MAT-loaded MIPs were validated through light microscopy analyses, and the matrine content in culture medium was quantified using ultra high-performance liquid chromatography-mass spectrum synchronously. The facile fabrication of MAT@MIPs presents a viable solution for designing high-loading and pH-responsive DDS, which can offer a novel administration approach for drugs requiring injection in clinical applications.
1 Introduction
Matrine (MAT), a primary constituent of Sophora moorcroftianam, which has a variety of pharmacological effects and is often used to reduce scar tissue, inhibit proliferation of cancer cells, and protect against myocardial injury. It has been widely used as an injection in hepatitis and antitumors (1). At present, matrine injection is mainly used intravenously and intravenously infusion in clinical applications (2). Despite the inherent advantages of injections, such as prompt efficacy, reliable action, and long-term effect, their drawbacks cannot be overlooked, for example, stringent pyrogen testing progress, complex preparation, inconvenient application, and injection pain (3,4). The irreversible physiological effects and clinical accidents caused by injections underscore the necessity for innovative administration methods for matrine (5,6).
For this purpose, researchers pay attention to the nano drug delivery systems (DDS) made of nanotechnology for drugs and carrier materials. This approach allows drug encapsulation within nanomaterials or attachment to their surfaces or interiors, facilitating targeted drug delivery and controlled release under specific conditions (7,8). Molecularly imprinted polymers (MIPs) emerge as promising carriers in DDS due to their selective recognition absorption capabilities (9,10,11) and the controlled drug release (12,13). The imprinted sites in the MIP structure are cavities, which are formed during the polymerization process in the presence of the target molecule (template) and then extraction of this molecule from the polymer matrix. After the removal of the template, the resulted permanent cavities are complementary with the template in size, shape, and the functional groups, and this complementary leads to MIPs’ affinity toward the template molecule (14,15). Furthermore, the preparation process of MIPs is simple and exhibits high stability against harsh conditions. Due to the aforementioned advantages, MIPs show a great potential in chromatography, electrochemical sensors and biosensors, solid phase extraction, and also DDS (16,17,18,19).
Considering the diverse pH values in different human organs, tissues, and cells. The pH of tumorous and inflamed tissues lower than the pH of blood (pH 7.4) and normal tissues, which ranges from 5.5 to 7.0. Thus, the pH value could be a suitable stimulus for the controlled release of the drug. The term pH-responsive polymer refers to materials containing ionisable pendant residues such as weak acidic or basic groups that are able to make changes in the structure or properties of the polymer (such as chain conformation, solubility, or surface activity), and in this way, it replies to the environmental pH changes in a controlled manner, which could be used to obtain the high therapeutic efficacy in DDS (20,21). The pH-responsive MIPs have attracted increasing attention as smart systems in drug delivery (22,23).
In the present study, multiwalled carbon nanotubes (MWCNs) and methacrylic acid (MAA) were used as the carrier and functional monomer, respectively, and dopamine (DA) was used as a crossing linker to form an imprinted shell on the surface of MWCNTs.
The self-polymerization of DA, known for its excellent film-forming ability (polydopamine layer) under weak base conditions at room temperature, contributes to the biocompatibility, biodegradability, and low toxicity of polydopamine (PDA), making it an ideal material for drug delivery (24,25). By surface imprinting technology, a pH-responsive MAT DDS was produced via self-polymerization of DA on the surface of MWCNs. After characterization of MAT@MIPs through Fourier transform infrared spectrometry (FTIR), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA) and elemental analysis, we evaluated the adsorption kinetics and adsorption isotherms of MAT@MIPs nanocomposite, and the drug release of MAT-loaded MAT@MIPs was evaluated in different pH values in vitro. The results show that the matrine release amount of the MAT-loaded MIPs is significantly increased with a decrease in the pH value, demonstrating the pH-responsive release ability of the materials, which will improve the drug utilization ratio and enhance the therapeutic effect in tumor tissues. Finally, the biosafety and the tumor-killing effect of MAT-loaded MIPs were verified by cell counting kit-8 (CCK-8) assays on HepG2 cells; moreover, we observed that MAT-loaded MIPs had an obvious sustained and prolonged release function and its cell uptake ability was also improved. Considering the advantages of the proposed imprinting polymers in the DDS, it could potentially optimize of drug administration mode and develop new dosage forms.
2 Experimental
2.1 Materials
Matrine (MAT, 98%, powder) was obtained from Shanghai Aladdin Biochemical Co., Ltd. Carboxyl-functionalized MWCNs (outer diameter 20–30 nm, –COOH content 2.00 wt%, 95% of purity) was purchased from Beijing DK Nano Technology Co., Ltd. (China). Dopamine and methacrylic acid (MAA, 98%) were obtained from J&K Scientific Ltd. (China) and Sinopharm Chemical Regent Co., Ltd, respectively. Phosphate buffer solutions (PBSs) were meticulously prepared using NaH2PO4 and Na2HPO4 (Tianjing Damao Chemical Reagent Co., Ltd.) with deionized water. All other chemicals utilized were of analytical reagent grade and supplied by local vendors.
2.2 Characterization
The particle sizes and morphology of MAT@MIPs and nonimprinted polymers (NIPs) were observed with a Sigma-300 scanning electron microscope (SEM, Zeiss Co., Germany). Fourier transform infrared (FTIR) spectra were recorded by a Nicolet IS5 FTIR spectrophotometer (Thermo Fisher Co. USA) using the KBr pressing method. The elemental contents were conducted utilizing an automatic elemental analyzer (Elmentar-UNICUBE, Germany). The thermal stabilities of MAT@MIPs and NIPs were assessed through the thermogravimetric analysis (TG-DTA7200, Hitachi, Japan) under a nitrogen atmosphere at 20–800°C with a heating rate of 10°C·min−1.
2.3 Preparation of MAT@MIPs and NIPs
In this investigation, dry MWCNs particles (80 mg, as the carrier) and 10 mL of Tris-HCl (20 mmol) were added into a 50 mL Erlenmeyer flask for the preparation of imprinted polymers. Following a 5-min ultrasonic dispersion, MAA (33.9 μL) and MAT (25 mg) were added as functional monomer and template, respectively. After 2 h of preassembling under magnetic stirring at room temperature, dopamine acrylamide was added as a crosslinking agent and an initiator. The mixture was stirred at room temperature for an additional 6 h. Upon completion of the reaction, methanol was employed to eliminate unreacted reagents, and a CH3OH-HAc (4:1, v/v) was used to elute template molecules. Subsequently, polymers were neutralized through washing with distilled water (pH test paper) and dried under a vacuum, resulting in the formation of molecularly imprinted polymers (MAT@MIPs). The nonimprinted polymers (NIPs) were prepared by identical procedures, albeit without the inclusion of the template molecule (MAT) to serve as a control.
2.4 Adsorption experiments
In kinetics experiments, MAT@MIPs and NIPs (10 mg) were introduced into the Erlenmeyer flask with 5 mL of 100 μg·mL−1 matrine solution, and the mixture was shaken at 30°C. The content of residual matrine in the solutions was determined after 1–180 min adsorption by the UV-Vis spectrometer (220 nm, Shimaozu-UV1750).
In adsorption isotherms, MAT@MIPs and NIPs (10 mg) were added into 5 mL of MAT solutions with concentrations ranging from 10 to 200 μg·mL−1 (in water). After dispersion and stirring for 30 min, the sorbent was extracted, and the matrine concentration in the filtrate solution was measured at UV-Vis220 nm. Finally, the amount of matrine bounded to nanocomposites was calculated by using blow Eq. 1 (26). Consequently, the recognition performances of MAT@MIPs and NIPs were preliminarily evaluated based on the kinetic and isothermal adsorption data described earlier.
where C 0 and C e (mg·mL−1) represent the initial and equilibrium concentrations of MAT, respectively; V (mL) is the volume of the MAT standard solution; and m MIPs (g) is the mass of MAT@MIPs.
2.5 Release experiment
The release kinetics of matrine from polymers were systematically investigated through the following procedure: MAT-loaded MIPs (30 mg) were resuspended into 50 mL of PBS solution (pH 4.0, 5.0, 6.0, or 7.0) and incubated at room temperature (the process of forming MAT-loaded MIPs was illustrated in Figure S1). At a certain time interval, 5 mL of supernatant was withdrawn, and subsequently, another 5 mL of fresh PBS solution (same pH) was added immediately. The released matrine in PBS was measured by UV-Vis220 nm. Ultimately, the release percentage was calculated with Eq. 2 (27).
where W r is the total cumulative release percentage of matrine; C i and C n are the matrine concentrations (mg·mL−1) for the ith and nth replacements, respectively; V e (mL) is the volume of PBS removed (5 mL); V 0 is the total volume of PBS (50 mL); and m drug is the total mass of matrine encapsulated (mg).
2.6 Hemolysis assay
For the quantitative hemolysis assay of MAT and MAT-loaded MIPs, we employed a method based on the released hemoglobin from the erythrocytes, following the protocol outlined by Song et al. (27). In brief, fresh mouse blood was prepared in a tube and diluted 50-fold by saline to obtain 2% erythrocyte suspension. MAT and MAT-loaded MIPs (1 mM MAT equivalent) were added into 2% erythrocytes suspension separately. Following a 4 h incubation at 37°C, erythrocytes pellets were separated by centrifugation (2,000 rpm, 15 min). The absorbance of the supernatant at 400 nm was measured to test the absorbance of hemoglobin. Saline (negative group) and distilled water (positive group) were considered to represent 0 and 100% hemolysis, respectively.
2.7 Cell culture
HepG2 cells (purchased from ATCC, Manassas, VA, USA) were cultured in DMEM basic (1×) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Excell Bio, Shanghai, China) and penicillin/streptomycin. Cells were cultured with 5% CO2 in a 37°C incubator. Before use, the morphology of HepG2 cells was verified by comparing characteristics and images provided by ATCC, and the cells were cultured strictly according to the ATCC protocol. Experiments were initiated when cells reached 60–70% confluence.
2.8 Cell cytotoxicity
The cytotoxicity assay utilized the protocols for the cell counting kit-8 (CCK-8) test (MedChemExpress, Cat: #HY-K0301) to assess the viability of MAT-loaded MIPs and free matrine-treated cells. The absorbance of the samples was measured with an Multiskan-G0 (Thermo Scientific) at a wavelength of 490 nm. HepG2 cells were seeded in 96-well plates (1,000 cells/well) and incubated at 37°C overnight before treatment with MAT-loaded MIPs or free matrine. To assess the toxicity of the matrine, HepG2 cells were exposed to varying concentrations of matrine (10, 20, 40, 80, 160, and 320 μg·mL−1). Concurrently, HepG2 cells were treated with MAT-loaded MIPs at the same dose for the comparative analysis. All experiments were performed in triplicate, and after 48 h of drug treatment, cell viability was detected by the CCK-8 test.
2.9 In vitro cellular uptake
HepG2 cells were plated in 12-well plates with a cell density of 2 × 104 cells per well and incubated at 37°C for 24 h. Then 160 μg·mL−1 MAT and MAT-loaded MIPs were added to the wells and incubated for 48, 72, and 96 h at 37°C. The cell morphology was observed using an inverted biological microscope (OLYMPUS-CKX53). Following fixation with paraformaldehyde for 20 min, the cells were stained with crystal violet solution for 30 min (1,000 μg per well). After rinsing with pure water and air drying at room temperature, the cell morphology was observed and recorded. Furthermore, HepG2 cells were seeded in six-well plates with 1 × 105 cells in each well and treated with drug after 24 h. Each group included three re-wells. The cell culture medium was collected at 48, 72, and 96 h after administration and detected by ultra high-performance liquid chromatography-mass spectrum (UHPLC-MS) (the conditions were detailed in Supplement Materials).
3 Results and discussion
3.1 Synthesis of MAT@MIPs
As depicted in Figure 1, the synthetic process of MAT@MIPs consisted of the following three key steps. First, methacrylic acid and matrine pre-assembled under the action of hydrogen bonding on the surface of MWCNs-COOH (serving as the carrier); Second, a thin adherent polydopamine (PDA) layer, incorporating embedded matrine, was formed by a one-step spontaneous deposition of dopamine in a weak alkaline solution (20 mmol, pH 8.5 Tris-HCl) (28). The polymerization of MAA was initiated and crosslinked by dopamine, leveraging its oxidability to trigger free radical reactions (29). Finally, CH3OH-HAc (4:1, v-v) was used as the eluent to remove the embedded matrine, resulting in the creation of recognition cavities on the carrier surface that were complementary to the template. To enhance the recognition capability, we meticulously optimized the preparation conditions, including the ratio of template (MAT) and functional monomer (MAA), reaction time, and the pH values of porogen (Tris-HCl) during the preparation process.
Schematic illustration of the preparation process of MAT@MIPs.
3.1.1 Effect of the ratio of template and functional monomer
The impact of the mole ratio of MAT and MAA was systematically investigated across the range of 1:3, 1:4, and 1:5. As depicted in Figure 2a, an increase in the MAT-to-MAA ratio from 1:3 to 1:5 exhibited minimal variation in MAT@MIPs adsorption for matrine; however, NIPs demonstrated the lowest adsorption for matrine at the optimal ratio of 1:4, resulting in the highest imprinted factor (IF = 2.91, calculated as IF = QMIPs/QNIPs). This observed trend may be attributed to insufficient binding sites provided by a low amount of functional monomers (MAT and MAA) at a 1:3 ratio, leading to a reduced adsorption capacity and a lower imprinted factor (IF = 2.53). Conversely, a high ratio (1:5) led to excessive functional groups of MAA distributed randomly throughout the nonimprinted polymers (NIPs), resulting in higher nonspecific adsorption and a decrease in the imprinted factor (IF = 2.41) (30). Consequently, the selection of a 1:4 ratio yielded a moderate adsorption capacity and the highest imprinted factor.
Effect of the ratio of template and functional monomer (a), the pH values of porogen (b), and reaction time (c) on the adsorption performance of MAT@MIPs and NIPs.
3.1.2 Effect of the pH values of porogen
The effect of the pH values of porogen was further explored within the range of 5–10 (shown in Figure 2b). As previously mentioned, the spontaneous self-polymerization of dopamine occurs under weakly alkaline conditions. Dopamine possesses both amino and hydroxyl groups, and these functional groups can engage through multihydrogen bond and hydrophobic interaction with matrine, which is advantageous for obtaining high binding capacity and efficiency. The self-polymerization reaction was compromised under acidic or strong alkaline conditions, leading to alterations in the polydopamine (PDA) layer. These changes affect the surface interaction with the carrier and result in reduced adhesion.
3.1.3 Effect of reaction time
To maximize the formation of imprinting cavities and also minimize the generation of nonspecific interactions, the effect of the reaction time was thoroughly investigated finally. As shown in Figure 2c, MAT-MIPs exhibited the best binding amount and IF among all the tested polymers when the polymerization time was set at 6 h. This observation suggested that the best PDA layer thickness and the optimal recognition sites were achieved within this timeframe. Further prolonging the polymerization time could prompt higher adsorption amounts in NIPs, which resulted in a low imprinting factor. Consequently, the optimized preparation conditions were determined to be MAT:MAA (1:4), Tris-HCl (pH8.5), and a reaction time of 6 h.
3.2 Characterization
3.2.1 SEM
The size and morphological changes of the MAT@MIPs was observed through SEM and TEM images (Figure 3). Bare carrier exhibited a relatively smooth surface with lengths of tens of microns (Figure 3a). Following the coating process, MAT@MIPs (Figure 3b) exhibited noticeably rough surface when compared with bare MWCNs-COOH, indicating that the PDA film has been deposited on the nanotube surface successfully. Such a thin imprinted layer was conducive to the mass transfer process between the adsorbates and the imprinted sites, so as to promote the adsorption performance. On the other hand, NIPs (Figure 3c) had some slight adhesion among neighboring nanotubes, implying that the absence of matrine during the synthesis process would affect the uniformity of the PDA layer, which can provide a high mass transfer between the solution and the imprinted sites. In Figure 3d, the MAT-loaded MIPs presented a rougher and more tufted surface than that of imprinted polymers, indicating the successful loading with matrine. From TEM images shown in Figure 3e and f, it can be inferred that the diameter of MAT-loaded MIPs became larger than that of the naked MWCNTs (Figure 3f), which demonstrates the successful in situ spontaneous oxidative polymerization of dopamine on the surface of the carrier. From the SEM and TEM images, it could be inferred that the imprinted nanofilms were successfully introduced on the surface of MWCNTs.
SEM images of (a) MWCNs; (b) MAT@MIPs; (c) NIPs; (d) MAT-loaded MIPs; TEM images of (e) MAT-loaded MIPs and (f) MWCNs.
3.2.2 FTIR
Fourier transform infrared (FTIR) spectra provided a direct proof for the deposition of PDA on the surface of the carrier. The strong absorption peaks at about 3,430 cm−1 (O–H) and 1,098 cm−1 (C–O) were the characteristic peaks of the hydroxyl group in the MWCNTs-COOH (Figure 4a). The peaks at approximately 2,936 and 1,584 cm−1 could be attributed to the stretching vibration of the C–H and the carbon–nitrogen (C–N) bonding of the matrine. Following the coating with dopamine, the bands at both 1,506 cm−1 were attributed to the superposition of phenylic C═C stretching, indicating that the PDA layers were formatted on the carriers. Similar peaks appeared at 1,464.46, 1,094.41, and 1,579.35 cm−1 on the MAT-loaded MIPs, indicating successful encapsulation of matrine within the cavities of MAT@MIPs.
(a) Fourier transform infrared spectroscopy and (b) TGA results of MWCNs, MAT@MIPs, NIPs, and MAT-loaded MIPs.
3.2.3 TGA
The weight of organic layers on the prepared polymers was assessed through thermogravimetric analysis (TGA). As illustrated in Figure 4b, when the temperature was below 400°C, a relatively low proportion of mass loss was observed in MAT-loaded MIPs, which potentially attributed to the volatilization of surface-bound water. The result indicated that the thermostability of MAT-loaded MIPs could be for normal use. To assess the amount of PDA film attached to the MAT-MIP surface, TGA analysis was also carried out for bare MWCNs-COOH and MAT-MIPs. In the temperature range of 400–570°C, the mass loss ratio for MAT-loaded MIPs was approximately 47.10%, while MAT@MIPs, NIPs, and MWCNs-COOH showed lower weight losses, which were only about 11.03%, 11.86%, and 9.06%, respectively. This observation can be attributed to the degradation of the functional groups, such as –NH2 and –OH groups, along with the organic components in the imprinting layers. When the temperature continued to increase to 800°C, the weight loss of all the four particles was up to 96%.
3.2.4 Elemental analyses
Table 1 displays the elemental percentages of MWCNs, MAT@MIPs, MAT-loaded MIPs, and NIPs. The nitrogen element in MAT@MIPs, MAT-loaded MIPs, and NIPs were higher than that of in MWCNs, suggesting that DA was grown on the surface of MWCNs. Meanwhile, the content of carbon elements all increased in MAT@MIPs and NIPs, demonstrating that the imprinted polymers were successfully grafted onto the surface of MWCNs. In addition, there was 0.06% nitrogen content in the data of MWCNs, potentially attributed to impurities in the obtained MWCNs (95% purity).
The results of elementary analysis
| N (%) | C (%) | H (%) | |
|---|---|---|---|
| MWCNs | 0.06 | 94.03 | 0.48 |
| MIPs | 0.59 | 92.72 | 0.34 |
| NIPs | 0.47 | 94.44 | 0.30 |
| MAT-loaded MIPs | 0.60 | 94.34 | 0.36 |
3.3 Adsorption study
3.3.1 Isothermal adsorption
The isothermal adsorption curves of MAT@MIPs and NIPs are shown in Figure 5a. As the concentration of matrine solution increases, the loading capacity of both MAT@MIPs and NIPs increase continuously until a concentration of 100 μg·mL−1, reaching the adsorption amount platform of 21.48 mg·g−1, approximately 2.36 times (IF = 2.36) greater than that of NIPs. This outcome indicated the successful formation of specific recognition sites on MAT@MIPs that are complementary to matrine in terms of spatial structure, molecular size, and chemical effects. In contrast, the adsorption of NIPs is primarily nonspecific. Subsequently, the isotherm data were fitted by using the following Langmuir (Eq. 3) and Freundlich isotherm models (Eq. 4) according to Shu’s work (31).
Adsorption kinetics (a) and isotherms (b) of MAT@MIPs and NIPs.
where Q e (μg·mg−1) is the adsorption capacity at equilibrium; C e (μg·mL−1) is the equilibrium concentration of matrine; K L is the Langmuir constant; Q m is the maximum theoretical adsorption capacity; K F is the Freundlich constant; and n is the heterogeneity factor.
The relevant parameters are calculated and listed in Table S1. The adsorption process did not conform to the Langmuir equation, as evidenced by a W-shaped curve (data not shown), indicating non-single-layer adsorption. For both MAT@MIPs and NIPs, the correlation coefficient of the Freundlich equation was determined to be more satisfactory (R 2 = 0.9773, 0.9979 respectively), which is widely considered as multilayer adsorption with nonuniform energy. Therefore, MAT@MIPs show multilayer adsorption behavior, allowing more than one matrine molecule to be adsorbed on a single adsorption site. In reality, when the template (MAT) was under a low concentration condition, the adsorption was usually carried with few cases of single-layer or multilayer adsorption saturation. Hence, the application of the Freundlich adsorption model is the most widespread in isotherm equation. In this work, MAT adsorbed by MAT@MIPs may be generated by nonuniform surfaces with uneven adsorption heat distribution and not limited to single-layer coverage (31).
3.3.2 Dynamic adsorption
The saturation binding experiments were used to investigate the kinetic adsorption of MAT@MIPs and NIPs. As shown in Figure 5b, the absorption capacity of MAT@MIPs and NIPs for matrine increased with adsorption time until reaching equilibrium, while SMIPs showed a more rapidly adsorption process within 30 min to reach equilibrium, with the adsorption amount reaching 21.48 mg·g−1, which is twice more than that of NIPs. MAT@MIPs showed high binding capacity and rapid mass transfer rate, which is possibly due to the porous structure of MAT@MIPs offering a very large number of binding sites on its surface. Subsequently, the adsorption kinetics data were fitted by the pseudo-first-order model (Eq. 5) and the pseudo-second-order model (Eq. 6) as follows:
Q e (μg·mg−1) is the adsorption capacity at equilibrium; Q t (μg·mg−1) is the adsorption capacity at time t; and k 1 and k 2 are the adsorption rate constants in the pseudo-first-order equation and pseudo-second-order equation, respectively.
The relevant parameters of the two pseudo-kinetic models are fitted and calculated as shown in Table S2. The R 2 value of the pseudo-second-order kinetic model was 0.9375, while the data could not fit with the pseudo-first-order kinetic model. Actually, there was no ideal condition of a single factor, and in this work, the rate of adsorption was not solely determined by one factor. Therefore, it implied that the adsorption process was determined by the square value of the unoccupied adsorption vacancies on the adsorbent surface, and the adsorption process was controlled by the chemical adsorption mechanism. This type of chemical adsorption involves electron sharing or electron transfer between the adsorption site and MAT.
3.4 Drug release profiles of the MAT-loaded MIPs
To investigate the release of matrine from MAT-loaded MIPs under different acidic conditions, the MAT-loaded MIPs (30 mg) were exposed to PBS (50 mL) at pH 4.0, 5.0, 6.0, and 7.0 at 37°C. As shown in Figure 6, in the first 5 h, the matrine was released rapidly with a release rate exceeding 40% in all four groups, then reaching desorption equilibrium at 22 h gradually. At pH 4.0 and 5.0, the total matrine release was 83.73% and 65.32%, while the total matrine releases at pH 6.0 and 7.0 were 53.38% and 46.98%, respectively. Moreover, the amount of desorption decreased gradually with the increase of the pH value, which was proofing the hypothesis that the matrine release from MIPs was regulated by the variation of pH. Based on these results, the MAT-loaded MIPs may be internalized and distributed in the endosome or lysosomal compartment (pH 4.0–5.0) of the tumor site, which makes the pH-responsive release of matrine possible in DDS. Subsequently, the release kinetic data were fitted by the zero-order, first-order, and Higucci and Korsmeyer–Peppas models, which can be defined as Eqs. 5–8 according to the work of Song et al. (27).
where Q t and Q m represent the cumulative release of MAT at time t and infinite time, respectively. k o, k 1, k H, and k KP are the respective constants of zero-order, first-order, and Higuchi and Korsmeyer–Peppas kinetic models. n is the release coefficient, which is used to characterize the release mechanism.
In vitro MAT release profiles of MAT-loaded MIPs at pH 4.0, 5.0, 6.0, and 7.0.
As shown in Table S3, the kinetic release behavior of MAT-loaded MIPs was more fittable to the Korsmeyer–Peppas model (R 2 > 0.92) under different pH conditions. The values of release parameter “n” are less than 0.3, indicating that the mechanism of drug release was Fick diffusion, where the release mechanism has been dominated by diffusion. However, the correlation coefficient was still below 0.97, indicating that there may be some dissolution release mechanism.
3.5 In vitro cell viability test
To ensure the safety and nontoxicity of the MWCNs, the cell viability test was performed initially by CCK-8 assays on HepG2 cells (Figure 7a), and the cell viability was kept at 80% or more when the concentration was less than 7.5 mg·mL−1 compared to the control group. This result confirmed that MWCNs were almost nontoxic and had good clinical application value. Then, to assess the therapeutic efficacy of MAT-loaded MIPs, HepG2 cells were incubated with MAT-loaded MIPs for 24 h compared with the free matrine group (Figure 7b). As the matrine dosage increased, the cytotoxicity of matrine to HepG2 cells increased synchronously. However, MAT-loaded MIPs had stronger cell-killing effects than that of free matrine, and when the concentration of matrine was at 160 μg·mL−1, the cell viability decreased by more than three times, which showed MAT-loaded MIPs had the enhanced curative effect. The possible explanations were as follows: on the one hand, the MAT-loaded MIPs has a good slow-release capacity because of the force of the imprinted pores on MAT. On the other hand, the PDA coating on MAT-loaded MIPs serves as a protective layer, which also limit the outward diffusion of MAT (32). Both the control of drug-release behavior was a key to avoiding excessive or insufficient drug release and maintaining a certain therapeutic drug concentration at the tumor cell for a long time in the DDS (33), which made MAT-loaded MIPs more effective in the curing process.
In vitro cytotoxicity of MWCNs (a) and MAT-loaded MIPs in comparison with free MAT (b) against HepG2 cells; (c) quantitative hemolysis assay of MAT (a) and MAT-loaded MIPs (b); (d) the content of matrine in the culture medium by UHPLC-MS at different time periods.
The same conclusion could also be drawn from the light micrograph, which showed the intuitive cell morphology in different groups: the control (PBS), free matrine, and MAT-loaded MIPs. As shown in Figure 8, the morphology was significantly different in the three groups. MAT-loaded MIPs showed an apparent aggregation phenomenon and fewer living cells, indicating more effective against HepG2 cells, whereas the free matrine group changed only slightly at the same dosage. Simultaneously, the content of matrine in the culture medium was detected by UHPLC-MS (Figure 7d), and the matrine content in the MAT group was two times higher than that of the MAT-loaded MIPs group at different times, which means the dopamine functionalized MAT-loaded MIPs had the ability to slow down and control release. It is worth noting that the content of matrine was increased slightly from 24 to 96 h in the MAT-loaded MIPs group, and this might be caused by the continuous release from MAT-loaded MIPs. Moreover, the hemolysis assay presented in Figure 7c showed that the MAT-loaded MIPs had good hemocompatibility, which was safe to use in the DDS. Combined with the results of Figures 7 and 8, it proved the safety and the excellent therapeutic effect of MAT-loaded MIPs on the tumor cell.
Light micrograph of HepG2 cells treated with MAT and MAT-loaded MIPs at 24–96 h.
4 Conclusion
We prepared the imprinted polymer nanoparticles (MAT@MIPs) on the MWCN. After the optimization of the preparation procedure and the characterization, MAT@MIPs were utilized for adsorption and controlled release of matrine. Then the experimental results in HepG2 cells confirmed the tumor cell-targeting and pH-responsive release properties in the DDS. Furthermore, the MAT-loaded MIPs presented good biocompatibility in hemolysis assay, which was a key prerequisite for in vivo experiments and further clinical application. The innovative approach could serve as an alternative solution for designing high-loading DDS with multiwall carbon nanotubes and provides a fresh perspective of thinking for the mode of administration of the injection, so as to overcome the adverse reaction of matrine injection in clinical settings.
Acknowledgements
This work was supported by the scientific research funding of the First Affiliated Hospital of Guangdong Pharmaceutical University (KYQDJF202014), National Natural Science Foundation of the School of Clinical Pharmacy of Guangdong Pharmaceutical University (SCP2022-01), and the Guangdong Basic and Applied Basic Research Foundation (SL2022A04J00538). And the 2022 Science and Technology Innovation Project of Guangdong Medical Products Administration “Research and Application of Key Technology and Evaluation System of Pharmacovigilance” (No. 2022ZDZ06).
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Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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