A Tasquinomod-loaded dopamine-modified pH sensitive hydrogel is effective at inhibiting the proliferation of KRAS mutant lung cancer cells

Abstract

Hydrogels can maintain a high local drug concentration during treatments and may be useful to local targeting diseased areas. We propose a pH sensitive hydrogel consisting of poly-vinylpyrrolidone (PVP) and chitosan as a new treatment method for KRAS mutant lung cancer. Addition of dopamine improved the drug loading and release effects of this hydrogel. We demonstrate that Tasquinimod-loading of this dopamine-modified pH sensitive hydrogel is more effective than Tasquinimod alone for inhibiting the proliferation of KRAS mutant lung cancer cells. Combination of conventional drugs with hydrogels may thus provide a new treatment modality for lung cancer.

Introduction

More than 1.5 million people die from lung cancer worldwide each year.^1,2 A recent study suggests that a third of the patients with most prominent subtype of lung adenocarcinoma carry a KRAS mutation.^3–6 Current clinical treatment for KRAS mutant lung cancer is based around chemotherapy, but traditional chemotherapy has the several shortcomings, including adverse effects. Moreover, using chemotherapy, a high drug concentration in focal tumor tissues cannot be guaranteed and therefore frequent injections are required to maintain effective drug concentrations.^7–10 In order to address these problems and to improve treatment efficiency, we proposed a pH sensitive hydrogel system modified by dopamine and loaded with chemotherapeutic drugs.

Hydrogels are hydrophilic polymers with excellent in vitro and in vivo biocompatibility.^11–15 Current applications based on these properties range from cell culture to biological scaffolds and injection drug delivery.^16–19 Here, we designed a dopamine-modified pH sensitive hydrogel based on chitosan and polyvinylpyrrolidone (PVP) (Figure 1), which accelerates the release of drugs in the low pH environment of tumor cells.^20–23 The adhesion of the material is increased due to the addition of dopamine: the catechol structure of dopamine and the hydrogel material are easily oxidized to a quinone or semiquinone structure when combined, followed by Michael addition or Schiff base reaction after which amino or sulfhydryl groups in the hydrogel appear and adhesion and cohesion are enhanced. ²⁴ Therefore, during treatment, high local drug concentration at the tumor site can be maintained, the drug influx into the systemic circulation is reduced, and side effects of chemotherapy are reduced.^25–29 But at the same time, the application field of hydrogels is facing some challenges. For example, Christensen LH, Breiting VB and Lloyd AW, Faragher RG, etc. found that the long-term application of hydrogels will have a certain impact on normal cell activities in tissues. Therefore, while taking advantage of the sustained-release drug properties of hydrogels, we shorten the treatment time of hydrogels as soon as possible to reduce adverse effects.^30,31

Figure 1.

Schematic diagram of dopamine-modified pH sensitive hydrogel.

Materials and methods

Synthesis of materials

Synthesis of the hydrogel: 0.15 g chitosan, 15 mL secondary water, and 2 mL H⁺ (3 mol/L) were added to a 5 mL round bottom flask and stirred well. 0.13 g of polyvinylpyrrolidone were added, stir, and vacuumized. 0.2 mL of formaldehyde (37%) were added and the reaction was left at room temperature for 24 h.

Dopamine cross-linking: 0.050 g of dopamine were added to 10 mL of Tris-HCl solution (0.05 mol/L) and stirred to dissolve. 1.0 g of previously synthesized hydrogel was added to the solution, soaked at room temperature for 48 h, filtered, and dried for later use.

Swelling Property of Hydrogel: Equal volume of freeze-dried hydrogels (10 g) were immersed in the PBS (pH 7.45/6) at 37°C. A total of 80 min of testing was carried out, and the hydrogel was taken out and weighed every 10 min until the end of the 80-min experiment (n = 3). The swelling ratio was calculated by the following equation: Swelling ratio (%) = (W_t–W₀)/W₀ × 100%.

Where W_t represents the weights of swelling hydrogels, and W₀ is the initial weights of freeze-dried hydrogels.

Hemolysis rate experiments: 1 mL of prepared red blood cells (Cell density: 4.5 × 10⁷/mL) were mixed with 3.67 mL of physiological saline. The solution was centrifuged at 1500 rpm for 15 min. This process was repeated three times to isolate a supernatant free from red blood cells. Four types of hydrogels were mixed with 1 mL of normal saline. After the hydrogel was fully dissolved, add 100 μL of each solution to 1 mL of physiological saline to prepare the test solution. 0.9% NaCl solution (1 mL) was used as a negative control, and deionized water (1 mL) was used as a positive control. Add 100 μL of the prepared red blood cell suspension to each group and incubate for 1 h (37°C). Centrifuge the samples for 15 min (300 rpm) and add 100 μL of each sample to a 96-well plate. The microplate reader measures the result at 540 nm. The rate of hemolysis (%) was calculated as = (As–An)/(Ap–An) × 100%, where Ap represents the OD value of the positive control group, As and An are the OD values of the experimental group and the negative control group respectively.

Cytotoxicity tests: 10 mL human umbilical vein endothelial cells (HUVEC, Cell density: 1 × 10⁵/mL) were cultured in RPMI1640 medium and DMEM. All cells were maintained at 37°C in an atmosphere of 5% CO₂ and 95% humidity and culture media contained 10% FBS. Cells were incubated for 24 h then seeded into 96-well plates. The same quality of the four types of hydrogels were added to the corresponding medium, sterilize after filtration. Then 1 mg/mL sterile mother liquor was obtained. Add 100 μL of sterile mother liquor to each well and co-culture with the cells for 1, 3, and 5 days. Finally, a CCK-8 (Focus Bioscience, Shanghai) assay was used to quantify the cell viability, and the optical density (OD) cell viability (%) was measured by a spectrophotometer: cell viability (%) was measured as OD result/OD control × 100%.

Live-dead cell staining: we used Calcein-AM/PI double staining reagent for live-dead cell staining. First prepare the working solution needed for staining (2 μmol solution A, Calcein-AM; 8 μmol solution B, PI). Take 5 μL 16 mmol solution B stock solution and add it to 10 mL of PBS solution, well mix. Obtain 8 μmol solution B working solution. Add 5 μL 4 mmol solution A stock solution to the 10 mL solution B working solution from the previous step, and make sure to mix well. The obtained mixed working solution (2 μmol solution A, Calcein-AM; 8 μmol solution B, PI) can be directly used for staining cells. Working solution was added to the hydrogel cells of the four experimental groups, incubated for 30 min and then washed with 10 μL PBS. Observe under the microscope (emission wavelength: 490 nm).

Drug-loaded release of materials: pH sensitive and insensitive hydrogels loaded with Tasquinimod (10 μmol) were placed in two pH gradients of 10 mL PBS (5.5/7.35) and allowed to release for 16 days. Every 2 days, 100 μL of the solution from each group were drawn with a pipette, added to a 96-well plate, and 100 μL of PBS were added immediately after extraction to supplement the experimental solution. Results were quantified using a microplate reader (for each experimental group, we set up three identical samples. n = 3).

In vitro test of tumor suppressive effect of materials: The experimental components were four groups, including: dopamine modified pH sensitive hydrogel + 2.5 μmol Tasquinimod, dopamine modified pH sensitive hydrogel + 10 μmol Tasquinimod, 5 μmol Tasquinimod, and 10 μmol Tasquinimod. To measure the activity of tumor cells, four groups of hydrogel groups loaded with anti-tumor drug Tasquinimod were co-cultured with tumor cells in vitro (for each experimental group, we set up three identical samples. n = 3; and the number of cells in each sample: 8 × 10⁶).

Cell cycle tests: Cells were seeded in a six-well plate at 1.5 × 10⁶ cells per well and allowed to grow to logarithmic growth phase. After 50%–80% adherence, cells were changed to the drug-containing culture medium (three wells per treatment). After incubation for the corresponding time, flow cytometry was conducted with the number of cells analyzed per sample being approximately 10⁶.

Cell processing: Cells were treated with trypsin and neutralized with serum-containing medium, centrifuged at 2000 rpm for 5 min, resuspended and washed with pre-cooled PBS. The supernatant was carefully aspirated, leaving about 50 µL.

Cell fixation: Cells were fully resuspended in 1 mL PBS. The cell suspension was gently vortexed while slowly adding 3 mL of pre-cooled absolute ethanol to a final concentration of 75%, incubated at 4°C overnight (18–24 h), and stored at −20°C for 1 month prior to flow cytometry. Fixed cells were washed with pre-cooled PBS twice, centrifuging at 2000 rpm for 5 min, and resuspended in 200 µL PBS, resulting in a final volume of about 400 μL. To reduce cell loss, 1.5 mL centrifugation volumes were used. The bottom of the centrifuge was gently tapped to properly disperse the cells and avoid cell clumps. 20 μg/L RNase, were added to the resuspended cells and cells were incubated in a 37°C water bath for 30 min. 20 µL of propidium iodide (PI) were added to a final concentration of 50 μg/mL and samples were stained for 30 min at 4°C under exclusion of light. For flow cytometry, which was conducted within 24 h of staining, cells were mixed thoroughly and filtered through a 200-mesh filter. Red fluorescence was detected using an excitation wavelength of 488 nm. Light scattering was assessed the same time. The appropriate analysis software was used for cell DNA content analysis and light scattering analysis.

Apoptosis test: Cells were washed twice with cold PBS buffer and resuspended in 1× Binding Buffer to make a suspension of 1 × 10⁶ cells/mL. 100 μL of the cell suspension were added to a Falcon test tube, gently mixed, and placed in a dark place at room temperature (20°C–25°C) for 15 min. Cells were washed once with 1× Binding Buffer and the supernatant was removed. 0.5 μg of the SAv-FITC reagent were dissolved in 100 μL of 1× Binding Buffer, added to the tube and gently mixed. 5 μL PI were added and cells were stained at room temperature (20°C–25°C) under exclusion of light for 15 min. 400 μL of 1× Binding Buffer were added to each test tube and apoptosis was quantified on a flow cytometer within 1 h.

Results and discussion

Characterization of materials

We constructed the experimental hydrogel via chemical cross-linking. Digital photographs in Figure 2(a) visualize the brown color of the hydrogel with dopamine, which is obviously different from other transparent and white hydrogels. The hydrogel base is a pure PVP and chitosan hydrogel (Figure 2(a)). During synthesis, we quantified the time required for the hydrogel to be completely set and found that the formation time of the hydrogel was halved after addition of dopamine (Figure 2(b) and (c)). This suggests that the addition of dopamine results in faster crosslinking speed and means that the material can quickly set at the tumor growth site, preventing it from shifting to non-treatment sites.

Figure 2.

Characterization of hydrogels. (a) Digital pictures of each type of hydrogel. From top to bottom: PVP hydrogel, chitosan hydrogel, PVP + chitosan hydrogel, PVP + chitosan + Tasquinimod hydrogel, PVP + chitosan + dopamine hydrogel. (b) Picture of the PVP + chitosan + dopamine + Tasquinimod hydrogel. (c) Comparison of the formation time of each type of hydrogel, *p < 0.05. **p < 0.01. ***p < 0.001. (d) Release of the drug from the PVP + chitosan + dopamine hydrogel at different pH levels, *p < 0.05. **p < 0.01. ***p < 0.001. (e) Swelling ratio of PVP + chitosan + dopamine + Tasquinimod hydrogel. Data are presented as mean ± SD (n = 3).

Acidic pH improves the drug-release capacity of the hydrogel

A key test for assessing the performance of a hydrogel is quantifying its drug release capacities. We obtained the drug release curve of the material by measuring the OD value of the Tasquinomod using a microplate reader. We tested both neutral and acidic pH environments, the latter mimicking the microenvironment surrounding the tumor to reflect physiological conditions for the use of the material. The release profile was quantified during a 16-day continuous release process. This revealed that the drug release rate is relatively slow at neutral pH levels, with release of approximately 45% of the total loading by day 16. Conversely, in acidic pH levels, the drug release rate is accelerated and the total release is greater, reaching about 70% by day 16 (Figure 2(d)). These data suggest a certain pH sensitivity of the material and highlight that it can release drugs quickly and in large quantities in the tumor microenvironment which tend to have a low pH, but not in tissues with normal pH. We tested the swelling properties of the hydrogel. As shown in Figure 2(e), the swelling ability of hydrogel in acidic environment is worse than that under normal human pH. This shows the effect of acidic conditions on our hydrogels.

The hydrogel has no effect on normal cell proliferation

Biological compatibility testing is particularly important in settings of clinical application. We first tested the hemolysis rate of the materials, which showed that the hemolysis rate of the hydrogel was below 5% in all four experimental groups, suggesting that each group of materials exhibits biological safety (Supplemental Figure S1). Next, we conducted cytotoxicity experiments to further explore the safety of the materials. The four materials were mixed and cultured with human umbilical vein endothelial cells for five days and the cytotoxicity of the materials was assessed. The HUVEC survival rate of all groups was above 90% for all days, highlighting that the material exhibits good biocompatibility (Figure 3(a)). Finally, we performed live-dead cell staining and found that all four experimental materials showed excellent biocompatibility in human umbilical vein endothelial cells (Figure 3(b)). In summary, these data illustrate the biological safety of materials and support the clinical application of our materials.

Figure 3.

Biological safety tests. (a) Live-dead cell staining of the control group, the PVP-chitosan hydrogel and the PVP-chitosan-dopamine hydrogel. (b) The materials of each group were co-cultured with HUVEC cells for 1, 3, and 5 days and cell survival was quantified. Data are presented as mean ± SD (n = 3).

The proliferation of A549 cells is effectively inhibited through low-concentration Tasquinimod combined hydrogel

Tasquinimod effectively inhibits the proliferation of nasopharyngeal carcinoma, ³² but there are no reports on its suitability in KRAS mutant lung cancer. We therefore conducted a gradient test for four different drug concentrations of Tasquinimod (0, 2.5, 5, and 10 μmol, and assessed cytotoxicity and viability in treated A549 KRAS mutant lung cancer cells after 5 days. Tasquinimod at 2.5 µmol resulted in an inhibition of tumor viability greater than 30%, with higher concentrations exhibiting even more pronounced inhibitory effects (Figure 4(a); Supplemental Figure S2).

Figure 4.

In vitro drug treatment. (a) The effect of drugs under different concentration gradients on the survival rate and inhibition rate of lung cancer cells for five consecutive days. (b) Inhibitory rate and survival rates of lung cancer cells following 2.5 and 10 µmol of the drug under single administration or hydrogel loading administration, *p < 0.05. **p < 0.01. ***p < 0.001. (c and d, e) Clone formation rate and quantitative graphs, *p < 0.05. **p < 0.01. ***p < 0.001. Data are presented as mean ± SD (n = 3).

Compared with the traditional one-time injection method, the advantage of hydrogel lies in its sustained release: a superior treatment effect can be achieved by maintaining high drug concentrations in the local treatment environment. We therefore compared hydrogel sustained release to one-time administration in order to explore treatment efficacy. To assess the release properties of the hydrogel, we conducted tests using two gradients of 2.5 and 10 µmol. Using different concentrations of hydrogels and direct administration for treatment of KRAS mutant lung cancer cells in vitro, we found that the hydrogel offers a superior inhibitory effect than one-time administration and exhibits a higher inhibitory peak, which showed a significant inhibitory effect at 2.5 µmol but even more pronounced inhibitory effects with 10 µmol (Figure 4(b)). Moreover, we found that the survival rate of tumor cells in the hydrogel treatment group was lower than in the corresponding control group (Supplemental Figure S3). In addition, we conducted a clone formation experiment to compare the hydrogel drug delivery group with the direct one-time administration group. This revealed that the drug significantly inhibited cell cloning, and the inhibitory effect was more obvious in the hydrogel drug-loaded group (Figure 4(c)–(e)).

The cell cycle of A549 cells is effectively inhibited through low-concentration Tasquinimod combined hydrogel

Finally, we explored the cell cycle stages in each experimental group by flow cytometry. The ability of the cells to divide was weaker in the Tasquinimod group than in the control group. The cell cycle in the Tasquinimod group was elongated, and the G1 and S phases of the cell were altered (Figure 5(a) and (b)), which may be related to the mode of action of the drug.

Figure 5.

Exploration of the inhibitory effect of treatment. (a) Cell cycle analysis of the four groups. (b) Quantitative graphs of the results, *p < 0.05. **p < 0.01. ***p < 0.001. Data are presented as mean ± SD (n = 4).

Conclusions

The pH sensitive hydrogel developed by us is based on polyvinylpyrrolidone (PVP) and chitosan. Addition of dopamine to the hydrogel provides it with superior gel-forming and supporting properties: due to the addition of dopamine, the cross-linked hydrogel can set faster than the base hydrogel and load a larger amount of drugs without an impact on its pH sensitivity. The research in this report shows that the drug Tasquinimod can inhibit the proliferation of KRAS mutant lung cancer cells, and this effect is elevated when used in combination with the PVP-chitosan-dopamine pH responsive hydrogel system. This novel approach of treating KRAS mutant lung cancer through biological materials provides new avenues for clinical treatment with advantages such as elevated efficiency and fewer side effects.

Supplemental Material

sj-docx-1-jbf-10.1177_22808000211073729 – Supplemental material for A Tasquinomod-loaded dopamine-modified pH sensitive hydrogel is effective at inhibiting the proliferation of KRAS mutant lung cancer cells

Supplemental material, sj-docx-1-jbf-10.1177_22808000211073729 for A Tasquinomod-loaded dopamine-modified pH sensitive hydrogel is effective at inhibiting the proliferation of KRAS mutant lung cancer cells by Jun Xu, Chuxi Zhang, Chun Cheng, Jun Yang, Chenxi Li, Xia Liu and Yi Sang in Journal of Applied Biomaterials & Functional Materials