Cancer Therapy: pH-Responsive Curcumin Delivery Using L-Lysine Functionalized Mesoporous Silica Nanoparticles

Editor: Nina

Scientists synthesize L-lysine functionalized mesoporous silica nanoparticles for pH-responsive delivery of curcumin, enhancing targeted cancer therapy and improving therapeutic efficacy.

Key Preview

Research Question

The study investigates how L-lysine functionalized mesoporous silica nanoparticles (MS@Lys NPs) can serve as a pH-responsive drug delivery system for curcumin, an anticancer agent.

Research Design and Strategy

The research employs a systematic synthesis and characterization of MS@Lys NPs, followed by in vitro experiments to assess drug loading efficiency and pH-responsive drug release behavior.

Method

Mesoporous silica nanoparticles were first synthesized and then modified with L-lysine. The curcumin loading and release studies were conducted under varying pH conditions (7.4, 6.5, and 4.0).

Key Results

The study found that MS@Lys NPs achieved a curcumin loading efficiency of approximately 68% and exhibited significant drug release in acidic environments, suggesting a promising pH-responsive characteristic.

Significance of the Research

This research offers a novel approach to enhance the delivery of anticancer drugs, potentially improving therapeutic outcomes for cancer patients through targeted, stimuli-responsive drug delivery systems.

Introduction

Cancer remains one of the leading causes of morbidity and mortality worldwide, affecting millions of individuals each year. This complex group of diseases is characterized by the uncontrolled proliferation of abnormal cells that can invade and spread to other parts of the body. Despite advancements in early detection and treatment methodologies, the prognosis for many cancer patients remains poor due to the aggressiveness of the disease and its ability to develop resistance to conventional therapies. Current treatment options often include surgery, chemotherapy, and radiation therapy, each with its own set of challenges and limitations.

Traditional drug delivery methods in cancer treatment typically involve systemic administration of chemotherapeutic agents, which aim to target rapidly dividing cancer cells. However, these methods often lack specificity, leading to significant collateral damage to healthy tissues and cells. This non-specificity results in various adverse side effects, including nausea, hair loss, and immunosuppression, which can severely impact the quality of life for patients undergoing treatment. Furthermore, the effectiveness of chemotherapy can be hindered by the development of drug resistance, where cancer cells adapt and become less susceptible to the therapeutic agents, necessitating higher doses that further exacerbate side effects.

In light of these challenges, there is a pressing need for innovative drug delivery strategies that can enhance the efficacy and safety of cancer therapies. One such approach is the development of stimuli-responsive drug delivery systems, which leverage specific physiological conditions—such as pH, temperature, or the presence of certain enzymes—to release therapeutic agents in a controlled manner. This strategy aims to improve the targeting of drugs to cancerous tissues while minimizing exposure to healthy cells, thereby reducing side effects and improving overall therapeutic outcomes.

This study explores the synthesis and characterization of pH-responsive mesoporous silica nanoparticles (MS@Lys NPs) functionalized with L-lysine, which serve as a novel drug delivery system for curcumin, an anticancer agent known for its potential therapeutic benefits. By harnessing the unique properties of these nanoparticles, this innovative approach seeks to address the shortcomings of traditional drug delivery methods, ultimately improving treatment efficacy and patient quality of life in cancer therapy.

Research Team and Aim

The research was conducted by a collaborative team of scientists, led by Madhappan Santhamoorthy, who spearheaded the investigation into innovative drug delivery systems for cancer therapy. The study was conducted in 2023 at the School of Chemical Engineering, Yeungnam University, Republic of Korea. The findings of this research were published in the paper titled “L-lysine Functionalized Mesoporous Silica Hybrid Nanoparticles for pH-Responsive Delivery of Curcumin” in the journal Pharmaceutics.

The primary aim of the research, as articulated by the lead researcher, Madhappan Santhamoorthy, was to “synthesize L-lysine modified mesoporous silica nanoparticles (MS@Lys NPs) and evaluate their pH-responsive capabilities for curcumin delivery in cancer therapy.” This aim underscores the team’s focus on developing a novel drug delivery system that can enhance the therapeutic efficacy of curcumin while addressing the limitations associated with traditional chemotherapy methods.

Experimental Process

Experiment 1: Synthesis of Mesoporous Silica Nanoparticles (MS@GPTS NPs)

Primary Technique: The primary technique employed in this experiment is the sol-gel hydrolysis and co-condensation method for synthesizing mesoporous silica nanoparticles.

Key Steps:

1. Preparation of CTAB Solution: Dissolve 1.5 g of hexadecyltrimethylammonium bromide (CTAB) in 200 mL of deionized water, stirring magnetically until fully dissolved.
2. Addition of Ammonia: Introduce 3 g of ammonia solution to the CTAB mixture and stir for 30 minutes to obtain a clear solution.
3. Incorporation of Silica Precursors: Slowly add a premixed solution of 20:80 mol% of 3-glycidoxypropyl trimethoxy silane (GPTS) and tetraethyl orthosilicate (TEOS) in 5 mL ethanol to the CTAB solution while stirring.
4. Hydrothermal Treatment: Maintain the stirring for 12 hours at 45 °C followed by 24 hours at 80 °C.
5. Extraction of the Surfactant: Wash the resulting precipitate with deionized water and ethanol, then dry at 60 °C. Extract the CTAB using an ethanolic ammonium nitrate solution (1 g NH4NO3 in 50 mL ethanol) three times.

Data Collection and Analysis: The synthesized MS@GPTS NPs were characterized using techniques including X-ray diffraction (XRD) to confirm the mesoporous structure, and nitrogen adsorption-desorption studies to analyze surface area and pore volume.

Figure 1. (a) Low-angle XRD patterns

Result: The synthesized MS@GPTS NPs exhibited a specific surface area of 642 m²/g, a pore size of 3.6 nm, and a mesopore volume of 0.042 cm³/g, indicating successful synthesis of mesoporous silica nanoparticles.

Novel Aspect: This experiment highlights an efficient method of synthesizing mesoporous silica nanoparticles with a well-defined structure, which is critical for enhancing drug loading capabilities compared to traditional silica nanoparticles.

Experiment 2: Functionalization with L-Lysine (MS@Lys NPs)

Primary Technique: The primary technique used in this experiment involves surface modification through a ring-opening reaction.

Key Steps:

1. Preparation of L-Lysine Solution: Dissolve 0.2 g (0.0136 mol) of L-lysine in 10 mL of deionized water.
2. Dispersion of MS@GPTS NPs: Disperse 0.5 g of MS@GPTS NPs in 100 mL of an ethanol-water mixture (40:60) and sonicate for 5 minutes to create a homogeneous dispersion.
3. Reaction: While stirring, add the L-lysine solution to the MS@GPTS dispersion and allow the reaction to proceed for 12 hours at 70 °C.
4. Washing and Drying: Filter the resulting mixture, rinse with deionized water and ethanol, and vacuum dry overnight at 50 °C.

Data Collection and Analysis: The functionalization of L-lysine onto the silica surface was confirmed through Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA), which were used to assess the presence of amine and carboxylic acid functional groups.

Figure 2. (b) FTIR spectra of MS@GPTS NPs and MS@Lys NPs, respectively. © FTIR spectrum of pure L-lysine monomer.

Result: The MS@Lys NPs displayed characteristic FTIR peaks indicating successful functionalization, with TGA revealing approximately 9.1 wt.% of L-lysine attached to the nanoparticles.

Novel Aspect: This step introduced L-lysine as a dual-functional agent that enhances drug interaction sites, significantly improving the drug loading efficiency compared to traditional silica nanoparticles without functionalization.

Experiment 3: Drug Loading with Curcumin (MS@Lys/Cur NPs)

Primary Technique: The key technique for this experiment is the adsorption method for drug loading.

Key Steps:

1. Preparation of Curcumin Solution: Dissolve 50 mg of curcumin in 5 mL of ethanol.
2. Loading Process: Add 0.2 g of MS@Lys NPs to a round-bottom flask containing 50 mL of ethanol and the curcumin solution, then sonicate for 5 minutes and stir magnetically for 2 hours.
3. Centrifugation and Drying: Centrifuge the mixture, wash with ethanol, and vacuum dry the resulting curcumin-loaded sample overnight at 40 °C.

Data Collection and Analysis: Drug loading efficiency was calculated using UV-vis spectrophotometry by measuring the initial and remaining concentrations of curcumin after loading.

Scheme 1.Schematic representation for the preparation of MS@GPTS NPs (step-1); surfactant extraction and L-lysine functionalities modified MS@Lys NPs (step-2,3); curcumin loading into the MS@Lys NPs (step-4); pH-responsive drug release behavior of the MS@Lys/Cur NPs NPs (step-5), respectively.

Result: The MS@Lys/Cur NPs achieved a curcumin loading efficiency of approximately 68%, demonstrating a significant increase compared to the MS@GPTS NPs, which had a loading efficiency of about 32%.

Novel Aspect: This experiment underscores the effectiveness of L-lysine functionalization in enhancing the loading capacity of curcumin, showcasing a direct improvement over standard methods that lack such functional groups.

Experiment 4: pH-Responsive Drug Release Studies

Primary Technique: The primary technique utilized in this experiment is the dialysis bag method for evaluating drug release under varying pH conditions.

Key Steps:

1. Preparation of Release Medium: Set up phosphate-buffered saline (PBS) solutions at pH levels of 7.4, 6.5, and 4.0.
2. Dialysis Setup: Place 50 mg of curcumin-loaded MS@Lys/Cur NPs in a dialysis bag with a molecular weight cutoff of 10 kDa and immerse it in 10 mL of the PBS solution.
3. Sampling: At predetermined intervals, withdraw 1 mL of the release medium and replace it with an equal volume of fresh PBS to maintain sink conditions.
4. Analysis: Measure the concentration of released curcumin using UV-vis spectrophotometry at 435 nm.

Data Collection and Analysis: Cumulative drug release percentages were calculated and plotted against time for each pH condition to visualize the release kinetics.

Figure 3. (a) In vitro drug release from the (a) MS@GPTS/Cur NPs; and (b) MS@Lys/Cur NPs, respectively, at different pH conditions. © In vitro release behavior of free Cur at different pH conditions. (d) pH-induced Cur release behavior from the MS@Lys/CurNPs.

Result: The MS@Lys/Cur NPs exhibited approximately 20% release at pH 7.4, 65% at pH 6.5, and complete release at pH 4.0 over 48 hours, demonstrating significant pH-dependent release behavior.

Novel Aspect: This experiment highlights the pH-responsive nature of the MS@Lys NPs, allowing for controlled release in acidic environments typical of tumor tissues, which is a marked improvement over traditional drug delivery systems that lack such responsive features.

Experiment 5: In Vitro Cytotoxicity and Cell Uptake Studies

Primary Technique: The primary technique for these experiments includes the MTT assay for cytotoxicity assessment and fluorescence microscopy for cell uptake analysis.

Key Steps:

1. Cytotoxicity Testing: Seed MDA-MB-231 cells in 96-well plates and treat them with varying concentrations (25 to 200 µg/mL) of MS@Lys/Cur NPs and free curcumin for 24 hours.
2. MTT Assay Procedure: Replace the medium with MTT solution (5 mg/mL) and incubate for an additional 5 hours, then dissolve the formazan crystals in DMSO and measure absorbance at 570 nm.
3. Cell Uptake Assessment: Treat MDA-MB-231 cells with curcumin-loaded MS@Lys NPs and incubate for 1 or 5 hours. Fix the cells with paraformaldehyde and analyze under a fluorescence microscope.

Data Collection and Analysis: Cell viability percentages were calculated for the MTT assay, while fluorescence intensity was quantified to assess the uptake of curcumin by the cells.

Figure 4. In vitro cytocompatibility of MDA-MB-231 cells exposed to (a) only control cells; (b) MS@GPTS NPs, and © MS@Lys NPs treated cells, respectively, at different sample concentrations. (**, highly significant p < 0.01).

Figure 5. (a) Fluorescence microscopy images of MDA-MB-231 cells treated with (a) only control MDA-MB-231 cells; (b) cells treated only MS@Lys NPs without Cur loading; © cells treated with MS@Lys/Cur NPs for 1 h incubation; (d) cells treated with MS@Lys/Cur NPs for 5 h incubation, respectively. Scale bar = 80 µm.

Result: MS@Lys/Cur NPs showed around 90% cytotoxicity against MDA-MB-231 cells at the highest concentration, with significant fluorescence indicating effective cellular uptake of curcumin.

Novel Aspect: This dual-experimental approach demonstrated not only the enhanced cytotoxic effects of curcumin when delivered via MS@Lys NPs but also confirmed their capacity for effective internalization into cancer cells, showcasing a more effective strategy for targeted cancer therapy compared to conventional delivery methods.

Overall, these experiments collectively illustrate a robust method for synthesizing, functionalizing, and applying mesoporous silica nanoparticles for effective drug delivery, with significant improve

Conclusion

The successful development of the L-lysine functionalized mesoporous silica nanoparticles (MS@Lys NPs) as a pH-responsive drug delivery system for curcumin has been achieved through a systematic approach involving the synthesis, characterization, and evaluation of the nanoparticles’ drug loading and release properties. The integration of L-lysine onto the silica surface not only enhanced the drug loading efficiency but also allowed for controlled release behavior in response to varying pH conditions, which is particularly advantageous for targeting acidic tumor microenvironments.

The highlight of the study is the demonstrated ability of MS@Lys NPs to encapsulate curcumin with an impressive loading efficiency of approximately 68% and to release the drug in a controlled manner, with about 65% release at pH 6.5 and complete release at pH 4.0. These results indicate that the MS@Lys NPs can effectively maintain drug stability in physiological pH while enabling targeted delivery in acidic conditions typical of cancerous tissues. Furthermore, the in vitro cytotoxicity studies revealed that the MS@Lys/Cur NPs exhibited enhanced cytotoxic effects against MDA-MB-231 cancer cells compared to free curcumin and control nanoparticles. Overall, this research provides a promising platform for the development of advanced drug delivery systems, potentially improving therapeutic outcomes in cancer treatment.

Reference

Santhamoorthy, Madhappan, et al. “L-Lysine Functionalized Mesoporous Silica Hybrid Nanoparticles for pH-Responsive Delivery of Curcumin.” Pharmaceutics, vol. 15, no. 6, 2023, p. 1631. MDPI, https://doi.org/10.3390/pharmaceutics15061631.