Efficient Drug Release from Liposomes Introduced Tetrazine Derivatives with Pyrimidine Rings Using Click Chemistry

Abstract

We developed a novel drug release method using a bioorthogonal inverse electron demand Diels–Alder reaction on liposomal membranes. Based on reports that replacing pyridine with pyrimidine in tetrazine derivatives improves the reaction rate with strained dienophiles, we investigated if liposomes with tetrazine derivatives containing pyrimidine rings efficiently release drugs via click chemistry. We synthesized and evaluated a tetrazine compound (Tz2) bearing a pyrimidine ring. The reaction rate constant of Tz2 with a norbornene (NB) derivative, 5-norbornenecarboxylic acid (NBCOOH), was higher than that of Tz1 with a pyridine ring. Liposomes containing the synthesized Tz2 (Tz2-liposomes) were prepared, and the reaction between Tz2 and NBCOOH on the liposomal membranes was confirmed using high-resolution mass spectrometry. We encapsulated indium-111-labeled diethylenetriaminepentaacetic acid ([¹¹¹In]In-DTPA) in liposomes as a model drug. The release of [¹¹¹In]In-DTPA from Tz2-liposomes was observed after the addition of NBCOOH, with release dependent on NBCOOH concentration. Moreover, release from Tz2-liposomes was significantly higher than that from Tz1-liposomes. These results suggested that tetrazine derivatives with pyrimidine rings efficiently released drugs, likely due to enhanced reaction rates. These findings would advance the development of controlled drug release methods using click chemistry.

Introduction

Drug delivery systems (DDS) enable drug delivery when and where needed. Liposomes are representative DDS carriers that can encapsulate both lipophilic and hydrophilic drugs and are used clinically for cancer treatment.^1,2) For liposomal formulations to exert potent therapeutic effects, they must be stable in the blood, accumulate in tumor tissues, and efficiently release the encapsulated drugs. To date, many controlled drug release methods have been reported, wherein drugs are specifically released from liposomes in tumor tissues by internal stimuli such as pH^3,4) and enzyme activity,⁵⁾ or external physical stimuli such as light⁶⁾ and heat.^7,8) However, the environment surrounding cancer tissues is not homogeneous, raising concerns regarding individual differences in the therapeutic effects of internal stimuli. Light-triggered drug release is limited to superficial lesions in terms of therapeutic effect due to low light penetration. Heat treatment can also damage normal tissues, complicating the targeted treatment of cancerous tissue alone.

In recent years, bio-orthogonal reactions that proceed rapidly and selectively within biological systems have garnered significant attention in drug discovery research. We developed a novel drug release system using the inverse electron demand Diels–Alder (IEDDA) reaction, a bio-orthogonal reaction between tetrazine (Tz) and norbornene (NB) derivatives.^9–11) The IEDDA reaction between 2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide (Tz1, Fig. 1) and NB derivatives on liposomal membranes alters the membrane properties of liposomes and enhances drug release. Increased drug release improves the therapeutic effect in tumor-bearing mice.¹¹⁾ However, further improvements in drug release remain a challenge. In this study, we focused on enhancing the rate of the IEDDA reaction between Tz and NB derivatives on liposomal membranes to improve drug release from liposomes. The rate of the IEDDA reaction is governed by the energy difference between the highest occupied molecular orbital and the lowest unoccupied molecular orbital of the two unsaturated components. The smaller the energy difference between these frontier molecular orbitals, the faster the reaction proceeds. The rate of the IEDDA reaction can be improved by decreasing the electron density on the diene and increasing the electron density on the dienophile.¹²⁾ For Tz derivatives, replacing the pyridine ring with a pyrimidine ring at position 6 of the Tz ring decreases the electron density of tetrazine (diene) and enhances the reaction rate with the NB derivative.^12–14) In this study, we prepared liposomes introduced 2-hexadecyl-N-(6-(6-(pyrimidin-2-yl)-1,2,4,5-tetrazin-3-yl)-pyridin-3-yl)octadecanamide (Tz2, Fig. 1) with a pyrimidine ring. We evaluated their drug release characteristics to investigate the impact of the enhanced reaction rate, resulting from the structural substitution of the pyridine ring to the pyrimidine ring in tetrazine derivatives, on drug release from Tz-liposomes.

Fig. 1. Chemical Structure of Tz Derivatives

Results and Discussion

We have developed a novel method to release drugs from liposomes using the IEDDA reaction. The IEDDA reaction between Tz1 and 5-norbornenecarboxylic acid (NBCOOH) on the liposomal membranes causes drug release, which enhances the therapeutic effect of Tz1-liposomes.¹¹⁾ However, improved drug release is required to enhance therapeutic effects. To overcome this problem, in this study, the effect of the chemical reaction rate on drug release from liposomes was investigated by modifying the chemical structure of Tz compounds.

Synthesis

As shown in Supplementary Chart S1, Tz2 was synthesized in three steps. The structures of 4 and Tz2 were identified by electrospray ionization (ESI)-MS, and ¹H- and ¹³C-NMR (Supplementary Figs. S1, S2).

Evaluation of the Reaction Rate Constants between Tz and NB Derivatives

The progress of the chemical reactions between Tz1 or Tz2 and NBCOOH was confirmed by a decrease in absorbance at approximately 530 nm, the characteristic absorption band of Tz. This decrease corresponded with the color of the reaction solution, which changed from pink to yellow. Notably, rapid decay was observed upon the addition of higher concentrations of NBCOOH (Fig. 2A). Plotting the observed rate constants against norbornene concentration showed a linear dependence (Fig. 2B). The reaction rate of the IEDDA reaction improves by decreasing the electron density on the diene (Tz derivatives) and increasing the electron density on the dienophile (NB derivatives).^12–14) In Tz derivatives, it has been reported that replacing the pyridine ring with a pyrimidine ring increases the reaction rate with NB derivatives by 3 to 4-fold.^12–14) Consistent with these reports, the reaction rate of Tz2 bearing a pyrimidine ring (k = 0.144) was approximately 4 times higher than that of Tz1 bearing a pyridine ring (k = 0.036) (Table 1). This could be due to a decrease in the electron density of tetrazine upon replacing the pyridine ring with a pyrimidine ring.

Fig. 2. Determination of the Second-Order Rate Constants (k) of Tz1 and Tz2

(A) The decay in the absorbance of tetrazine derivatives (Tz1: 535 nm, Tz2: 530 nm) when Tz and NBCOOH were reacted at 37°C. (B) Plot of observed rate constants against NBCOOH concentrations.

Table 1. The Second-Order Rate Constants (k) of Tz1 and Tz2 Reacted with NBCOOH

	k (M–1 s–1)
Tz1 + NBCOOH	0.036 ± 0.002
Tz2 + NBCOOH	0.144 ± 0.001

Preparation and Characterization of Liposomes

Liposomes composed of Tz1 or Tz2, hydrogenated soybean phosphatidylcholine (HSPC), 1,2-distearoyl-sn-glycero-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (DSPE-PEG), and cholesterol (Tz1- or Tz2-liposome) was prepared by thin-film hydration followed by sonication method. Ctrl-liposomes without tetrazine were also prepared. The particle size of the prepared liposomes was approximately 50–75 nm, and their zeta potentials were slightly negative. The physicochemical properties of liposomes were not significantly altered by labeling with ¹¹¹In (Supplementary Table S1). The liposomes were loaded with indium-111-labeled diethylenetriaminepentaacetic acid ([¹¹¹In]In-DTPA) using a remote-loading method with high radiolabeling efficiencies (approximately 90%) and purities (> 97%) (Supplementary Table S2).

To confirm the chemical reaction between Tz and NBCOOH, high-resolution (HR)MS analysis was performed after mixing Tz2-liposomes with NBCOOH and adding methanol. HRMS analysis after reacting Tz2-liposomes with NBCOOH indicated an IEDDA reaction between Tz and NBCOOH (HRMS: m/z Calcd for C₅₃H₈₄N₆O₃ [M + H]⁺ 853.66777. Found: 853.66699; Supplementary Fig. S3).

WST assays were performed to analyze the cytotoxicity of the Tz2-liposomes using RAW264 cells. No cytotoxicity was observed for Ctrl- and Tz2-liposomes at any concentration (Supplementary Fig. S4).

In Vitro Stability of Tz2-Liposomes

To deliver the loaded drugs to the target tissues thorough the blood, the stability of liposomes in the blood is important. The stability of Tz2-liposomes in phosphate-buffered saline (PBS) and plasma gradually decreased with time, although the stability was over 83% at 48 h after incubation at 37 °C (Supplementary Fig. S5). The decrease in the stability of Tz2-liposomes might be derived from the instability of Tz2 because Tz compounds with stronger electron-withdrawing groups showed lower stability in an aqueous environment.^15,16) For the development of future drug release methods using the IEDDA reaction, it is necessary to design tetrazine derivatives that are stable in blood and have high drug release properties.

In Vitro Evaluation of Drug Release from the Liposomes

The release of [¹¹¹In]In-DTPA from the Ctrl- and Tz2-liposomes 24 h after the addition of various concentrations of NBCOOH is shown in Fig. 3A. A higher release from Tz2-liposome was observed with increasing concentrations of NBCOOH. The release from ¹¹¹In-labeled liposomes with PBS or 40 mM NBCOOH at 0.5, 3, and 24 h is shown in Fig. 3B and Supplementary Table S3. Tz-liposomes showed higher release than Ctrl-liposomes. Especially, Tz2-liposomes showed a significantly higher release than Tz1-liposomes at 3 and 24 h after the addition of NBCOOH. Because drug release was observed at an early time point (3 h), the increased reaction rate on the liposomal membranes may contribute to a more potent therapeutic effect of the liposomal formulations. In contrast, we found that NBCOOH showed higher drug release than some NB derivatives.¹⁰⁾ Although the carboxyl group is electron-withdrawing, NBCOOH showed a higher drug release than 2-NB in the reaction with Tz-liposomes, indicating that both electronic and steric differences may affect drug release. Modifying the structure of NB derivatives is an effective strategy for improving drug release. However, even a slight structural change in NB derivatives may have a large impact on their pharmacokinetics in the body, directly affecting chemical reactions and drug release in the tumor. Therefore, the chemical design replacing the pyridine ring with a pyrimidine ring in Tz compounds would be an effective strategy for increasing the reaction rate without significantly changing the structure of the compounds. The enhanced drug release from the Tz2-liposomes demonstrated the effectiveness of increasing the chemical reaction rate to develop a controlled drug release method from liposomes using the IEDDA reaction.

Fig. 3. In Vitro Release Test

(A) Release of [¹¹¹In]In-DTPA (%) from Ctrl- and Tz2-liposomes at 24 h after incubation in PBS or NBCOOH solution (20, 30, 40, and 60 mM). Results are expressed as mean ± S.D. (n = 3–4). Significance was determined using an unpaired t-test. *p < 0.05 vs. Ctrl-liposome. (B) Release of [¹¹¹In]In-DTPA (%) from Ctrl-, Tz1-, and Tz2-liposomes at 0, 5, 3, and 24 h after incubation in NBCOOH (40 mM). Results are expressed as mean ± S.D. (n = 3).

Conclusion

In this study, Tz2 bearing a pyrimidine ring was synthesized and demonstrated that a second-order rate constant (k) with NBCOOH was approximately 4 times higher than that of Tz1 bearing a pyridine ring. Tz-liposomes were prepared and labeled with ¹¹¹In by encapsulating [¹¹¹In]In-DTPA as a model drug to evaluate the effect of chemical reaction rate on drug release. The physicochemical properties of Tz2-liposomes were similar to those of Tz1-liposomes, probably because of the slight structural changes from Tz1 to Tz2. The release of [¹¹¹In]In-DTPA from the Tz2-liposomes was significantly greater than that from the Tz1-liposomes. These findings suggest that enhancing the reaction rate on the liposomal membranes can increase drug release without significantly altering the Tz compound structure, potentially leading to improved therapeutic efficacy.

Experimental

Synthesis of 2-Hexadecyl-N-(6-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)pyridine-3-yl)octadecanamide (Tz2)

Tz2 was synthesized in 3 steps. Detailed synthetic methods were described in Supplementary Materials.

Determination of Reaction Rate Constants

An equal volume of 1.5 mM solution of Tz derivatives (Tz1 or Tz2) and a solution of 5-norbornenecarboxylic acid (NBCOOH) (0.25, 0.5, 1, and 2 M for Tz1, 0.75, 1, 1.25, and 1.5 M for Tz2) in methanol were mixed, and the absorbance was measured immediately. Most tetrazines have a characteristic absorption band at around 540 nm.¹⁷⁾ The decay in the absorption of tetrazine derivatives (Tz1: 535 nm, Tz2: 530 nm) was followed over time at 37 °C. Each measurement was repeated 3 times, and the observed rate constants (k′) were plotted against the concentration of NBCOOH. The second-order rate constants (k) were obtained from the slope of the plot.

Preparation of Liposome with Tz Derivatives

Tz-liposomes were prepared by thin-film hydration followed by the sonication method as previously described.^10,11) HSPC, DSPE-PEG, cholesterol, and Tz derivatives (Tz1 or Tz2) were used (HSPC : DEPE-PEG : cholesterol : Tz derivative = 44.6 : 5 : 38 : 12.4 (mol%)).¹¹⁾ Control liposomes (Ctrl-liposomes) without Tz derivatives were prepared with HSPC : DEPE-PEG : cholesterol = 57 : 5 : 38 (mol%).

HRMS Spectra after the Reaction of Tz2-Liposomes with NBCOOH

The liposomes were diluted with PBS to achieve a final HSPC concentration of 0.2 mM. Tz2-liposomes were mixed with 80 mM NBCOOH at 1 : 3 (v/v) and allowed to react for 2.5 h at 37 °C. Methanol was then added to the reaction solution, and HRMS was performed immediately.

In Vitro Cytotoxicity of Tz2-Liposomes

RAW264 cells (1.0 × 10⁴ cells/well) were seeded into 96-well plates and incubated in a CO₂ incubator overnight. Then, Ctrl-liposome and Tz2-liposome (0.5–50 mM HSPC) were added to the RAW264 cells and incubated for 24 h. Cell viability was measured using a WST-8 assay (CCK-8).

In Vitro Stability Test of ¹¹¹In-Labeled Tz2-Liposomes

Liposomes were labeled with ¹¹¹In using the remote loading method as described previously.^18,19) The stability of Tz2-liposomes (HSPC concentration: 0.1 mM) in PBS or mouse plasma at 37 °C was assessed using size exclusion chromatography as described previously.¹⁸⁾ The stability of ¹¹¹In-labeled liposomes was calculated using the following equation:

Stability (%) = (radioactivity of [¹¹¹In]In-DTPA-encapsulated liposomes)/(total radioactivity added to the PD-10 column) × 100.

In Vitro Evaluation of Drug Release from the Liposomes

The release of [¹¹¹In]In-DTPA from liposomes upon adding different concentrations of NBCOOH was assessed using size exclusion chromatography. Briefly, NBCOOH (20, 30, 40, and 60 mM) or PBS was added to [¹¹¹In]In-DTPA-encapsulated liposomes (concentration of HSPC: 0.1 mM) at 3 : 1 ratio (v/v) and incubated at 37 °C. After 24 h, the solutions were added to the PD-10 columns and separated into fractions containing free [¹¹¹In]In-DTPA and [¹¹¹In]In-DTPA-encapsulated liposomes. The release of [¹¹¹In]In-DTPA was calculated as follows:

Release (%) = (radioactivity of free [¹¹¹In]In-DTPA)/(total radioactivity added to the PD-10 column) × 100.

The time-dependent release was examined by adding a 40 mM NBCOOH or PBS solution to [¹¹¹In]In-DTPA-encapsulated liposomes at 3 : 1 ratio (v/v). The samples were mixed well and incubated at 37 °C for 0.5, 3, or 24 h. The release of [¹¹¹In]In-DTPA from liposomes was calculated as described above.

Acknowledgments

This study was supported by JSPS KAKENHI [Grant number: 22H03030 (T. M.)]. The authors thank Dr. Atsuko Takeuchi for assistance with the HRMS measurements.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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