Iontronic click-to-release enables electrically controlled delivery of drugs and biomolecules beyond charge and size limitations

Domain: physics
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Dynamic and programmable control of therapeutic delivery is a long-standing goal in medicine. Iontronic devices offer precise electronic control over the dosage of bioactive molecules, yet their use has been confined to charged, low-molecular-weight compounds that are electrochemically stable during transport. Here, we present a hybrid delivery platform that integrates iontronic transport with bioorthogonal click-to-release chemistry. In this system, iontronic pumps electrophoretically deliver charged tetrazines as molecular scissors that selectively react with immobilized trans-cyclooctene (TCO)-linked payloads, enabling on-demand bioorthogonal cleavage of the TCO linker and controlled payload release. This approach retains the electronic precision of iontronics while overcoming molecular size, charge, and stability constraints. We demonstrate tunable tetrazine delivery over several days and electronically controlled release of immobilized payloads from small bioactive molecules, such as the antimitotic agent CA-4, to the large protein bovine serum albumin. Hence, by integrating bioorthogonal click-to-release strategies, iontronic delivery is extended to biologically relevant macromolecules, providing a foundation for advanced programmable electroceutical devices.

Introduction
Modern medicine increasingly recognizes that therapeutic efficacy depends not only on what drug is delivered, but also where, when, and how it is administered. The ideal therapeutic platform would achieve precise spatiotemporal control over drug dosing, dynamically adjusting to physiological needs, minimizing systemic exposure, and enabling personalized treatment profiles.1,2 Particularly for applications inside the human body, locally restricted release of drugs using different types of hydrogels3, nanocarriers4, and stents5 has gained considerable attention in the last years, showcasing an increased therapeutic effect while minimizing adverse systemic effects. However, current therapeutic implants lack real-time adaptability and offer limited control over dose timing.6-9 Put simply, once the drug release has been initiated, it cannot be turned off or adjusted. Many of these systems also suffer from the burst release effect10,11, characterized by an initial rapid release of a large drug fraction before switching to a constant passive release profile (red trace in Fig. 1). The inability to reprogram or modulate therapy by adjusting drug delivery profiles in situ compromises their therapeutic safety and limits their clinical impact.12 Implantable devices and osmotic pumps have been successfully designed to deliver drugs locally 4,13-20, but these technologies typically rely on pre-programmed release or external triggers. Such triggers can be chemical (e.g., pH changes/hydrolysis21,22, enzyme mediated cleavage23,24, oxidative stress/ROS25-27) or physical (e.g., magnetism28-30, temperature31, ultrasound32,33 or light34-36). The latter enable remote activation, converting externally applied energy into localized effects such as heating, mechanical stress, or photon-induced activation at the target site. Depending on the trigger platform used, this may require implantable release reservoirs or local administration of stimulus-responsive carriers (e.g., nanoparticles) that can be subsequently activated in situ. As activation relies on energy propagation through biological tissue, achieving precise spatial targeting and accurate dose control remains challenging. In addition, such energy transfer can adversely affect surrounding tissue and, for light-based systems, typically requires optical access to the target region. Iontronic pumps (IP) offer an alternative to several of these limitations by converting electrical signals directly into molecular transport, providing tunable delivery rates with high spatiotemporal control. IPs consist of (i) a source (drug) reservoir, (ii) an ion exchange membrane (IEM) forming the “channel” providing selective transport of charged species from the reservoir to the target region, and (iii) electrodes in reservoir and target regions.37,38 By applying a defined potential difference between source and target electrodes, species with opposite charge to the fixed charges in the IEM are electrophoretically transported (“pumped”) at a specific rate across the IEM into the target site without any liquid flow or the need for mechanical components. This enables the system to translate electrical signals into precise and customizable biochemical concentration-time profiles. IPs have been successfully used to control the delivery of small molecules such as neurotransmitters39-41, plant hormones42,43, modulators of inflammation44,45, and chemotherapeutics46-48. However, to ensure effective charge selectivity through the IEM (and thereby dose precision), the effective pore size in the hydrated IEM material limits IP-delivery to charged (ionic) compounds with low molecular weight (typically ≲600 Da) which are (electro)-chemically stable. Consequently, these restrictions severely limit the scope of therapeutics that are compatible with iontronic transport.49

To overcome these limitations, we combined iontronic delivery with subsequent click-to-release (C2R) chemistry to effectively decouple the transport mechanism from payload liberation or drug activation. C2R reactions are a class of bioorthogonal reactions in which a chemical trigger selectively reacts with a latent payload-bearing molecule, resulting in the initial formation of a covalent intermediate “click” product that undergoes rapid internal rearrangement “to release” the payload.50 This concept has previously been applied for targeted drug activation in vivo, including in hydrogel depot systems and antibody-drug conjugates.51-54 Although these approaches offer localized therapeutic action, they depend on systemic dosing of prodrugs and afford only limited control over the timing of activation. In contrast, the approach presented here demonstrates electronically controlled activation and enables tunable dosing. To implement this concept, we employed the click-triggered bond-cleavage chemistry of trans-cyclooctenes (TCO) and tetrazines (Tz), known for its exceptional kinetics and proven compatibility with biological systems.55 In particular, aminoethyl-functionalized Tz were shown to be efficient reagents56 to accelerate the click-to-release of cleavable TCO and, due to their permanent charge under physiological conditions, compatible with IPs. We therefore used symmetric aminoethyl tetrazine (1, Fig. 2c) as chemical trigger which was sufficiently small to be transported through the IEM (<200 Da) while offering the required high solubility and reactivity properties. Payloads were immobilized onto bead-scaffolds, which essentially served as on-demand activatable drug reservoirs via click‐cleavable TCO linkers (cTCO, Fig. 1). This allowed for payload modifications without being constrained by the charge or molecular weight limitations inherent to iontronic delivery. We initially demonstrated the controlled, programmable iontronic transport of Tz 1 and later showcased the controlled release of payloads using both the small-molecule chemotherapeutic Combretastatin A-4 (CA4, 316 Da) and bovine serum albumin (BSA, 66 kDa) from magnetic beads.

Results
Controlled Iontronic Tetrazine Delivery
Building on designs and results of controlled compound delivery using established capillary IPs43,57, we aimed to establish reliable Tz 1 delivery. We initially confirmed that aminoethyl tetrazine (1) remained intact under iontronic delivery conditions by cyclic voltammetry (Supplementary Information 9.2). The polymeric matrix enabling iontronic delivery of positively charged 1 was formed as negatively charged sulfonated cation-exchange membrane (CEM) within polyimide-coated fused silica capillaries (Fig. 2a) by UV-polymerizing 2-acrylamido-2-methylpropane sulfonic acid (AMPSA), poly(ethylene glycol) diacrylate (PEGDA, Mn 575), and photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP).58 All capillary IPs were fabricated based on previously reported procedures with improved custom-made Eppendorf or well-plate compatible 3D-printed housings (Fig. 2b, Fig. 4b, and Supplementary Information 4).57

IP performance was initially tested with the small cation K+ (0.1 M KCl) in the source before switching to Tz loading and subsequent delivery (Supplementary Information 5). The representative chronopotentiometry shows a stable low-voltage (~1 V) operation when a constant current (nanoamperes) was applied to the system (Supplementary Fig. S6). The amount of delivered 1 was determined using a fluorogenic assay via the C2R of 4-methylumbelliferone (4MU) from click-cleavable TCO-tag rTCO-DMEDA-4MU (2) and enabled quantification of 1 down to micromolar concentrations. An increase in applied charge (current × time) could be directly correlated to the amount of delivered 1, demonstrating full control over the iontronic transport (Fig. 2c) with an electrochemical transport efficiency of approximately 68% at +20 nA.

To validate the programmability and reliability of the transport, we operated devices for 2.5 days with alternating “ON” and “OFF” phases (16 h vs. 7 h, Fig. 2d) showcasing the stability, necessary for combining iontronic transport with the activation of (highly) potent drugs. Decreasing the applied current from +20 to +5 nA was shown to follow a linear trend. Delivery rates were 5.4 ± 0.7, 2.7 ± 0.4, and 0.7 ± 0.6 pmol min-1 for a constant applied current of +20, +10, and +5 nA, respectively (Fig. 2e). Short reversal of the current (-20 nA for 1 h) followed by removal of the active bias was shown to be enough to effectively halt passive diffusive processes through the membrane (Supplementary Fig. S9). Given an example target volume of 10 mL, these rates would correspond to an increase in the concentration of 1 on the order of ~0.5 nM min-1 during +20 nA active delivery. Considering the subsequent C2R kinetics (Supplementary Information 3.2), this release rate would enable efficient local release of highly potent drugs in the nM to µM concentration range over therapeutically relevant timescales of minutes to hours.

CA4 Prodrug Activation
To further prove the controllability and safety of the iontronic Tz pumping for drug activation, we tested the release of the highly potent anti-mitotic and anti-angiogenic agent Combretastatin A-4 (CA4, IC50 ~ 1 nM for HT1080 human fibrosarcoma cells).59 We used click-cleavable TCO-linker (cTCO)52, already used as caging moiety for CA4 and evaluated its C2R kinetic parameters with Tz 1. Second-order rate constant k2 was determined using stopped-flow kinetics between cTCO-(PEG2-OH) (3) and 1 to be 37 M-1 s-1 (Supplementary Fig. S1). The release process was studied by HPLC analysis using water soluble sulfo-cTCO-DMEDA-CA4 (4) prodrug with 1 (Fig. 3a) and was shown to be highly efficient, achieving near complete (>94%) CA4 release within 3 hours at micromolar concentrations (Supplementary Information 3.2). To confirm that release also occurs under iontronic conditions, we performed HPLC analysis during electronic delivery of 1 into a reservoir of prodrug 4. CA4 release increased proportionally with pumping duration (3-9 h), whereas K⁺ delivery did not induce any detectable release, confirming mechanistic fidelity and prodrug stability under iontronic operation (Supplementary Information 6.1).

Initial cell viability experiments with soluble CA4-prodrug 4 confirmed that 4 is non-toxic for the human glioblastoma cell line U-87 at nanomolar concentrations, but that a combination treatment of increasing concentrations of the prodrug 4 + 5 µM 1 showed comparable cell toxicity to parent CA4 (Fig. 3b) with a minimal cell viability of ~25%. Increasing CA4 concentration beyond this point did not lead to any additional decrease in viability, corresponding to the maximal toxic effect of CA4 in this system (Supplementary Information 6.2). Neither the TCO-bound prodrug 4 (up to 1 µM) concentration, nor Tz 1 (5 µM) affected the cells (99% cell viability). Both the released (4 co-incubated with 5 µM 1) and parent CA4 showed similarly reduced cell viability at 100 nM concentrations of 28% and 26%. In other words, the non-released prodrug 4 is effectively non-toxic until the release of CA4 is triggered by 1.

We therefore installed iontronic devices delivering 1 over cells in 100 nM 4 and operated the devices in forward (+) and reverse (-) constant current mode for 1 h at ± 20 nA (Fig. 3c, potential traces shown in Supplementary Fig. S12) followed by removal of the pumps and subsequent 72 h incubation. One device and its corresponding cell viability data was excluded due to reaching the maximum voltage of 10 V, indicating non-functional Tz delivery (Supplementary Fig. S13). Based on the established delivery rate of 1 at +20 nA, we estimated that active pumping for just 30 seconds would suffice to deliver ~10 nM of Tz 1 into 250 µL, thereby releasing a biologically relevant amount of CA4. While active delivery significantly lowered cell viability to 25%, correlating with the maximal toxic effect of CA4 in the U-87 cell line, the reverse operation effectively halted transport of 1 across the CEM and thus did not influence cell viability, exemplifying the high level of control achieved with iontronic delivery of the C2R trigger.

Iontronic Release of Immobilized CA4
Building on these results, we employed cTCO-bis-NHS (5) to synthesize biotin-cTCO-DMEDA-CA4 (6), enabling immobilization of the prodrug onto streptavidin-coated magnetic beads, which were selected as a well-defined solid support allowing quantitative analysis of release while serving as a simplified proof-of-principle model system. We used the discrepancy in reactivity of the NHS-carbonate and NHS-ester groups (Fig. 4a), which allowed for the sequential decoration of the cTCO-linker.52 This strategy facilitated the rapid synthesis of 6 using DMEDA, biotin-NH2, and subsequent reaction with CA4-p-nitrophenol (CA4-pNP) (Supplementary Information 2). The biotin moiety allowed 6 to attach onto magnetic streptavidin-coated beads (Dynabeads MyOneTM Streptavidin C1). Beads were co-incubated with U-87 cells in a transwell setup, and iontronic devices for delivery of 1 were positioned by immersing the device tip just above the beads within the transwell inserts (Fig. 4a, right). To facilitate consistent positioning of the iontronic devices within the 96-well transwell plates, a custom 3D-printed platform was developed (Fig. 4b). This setup accommodated the complex geometry of standard high throughput screening (HTS) transwell supports by incorporating a funnel-like mounting structure, offering precise placement of the device’s outlet in the top well and the counter electrode in the bottom compartment where the cells were cultured. The funnel serves as an agar bridge electrode and was filled with agarose hydrogel to maintain ionic contact while electrically isolating the electrode from direct contact with the cell culture environment, thereby reducing cytotoxic effects stemming from Ag/AgCl electrodes.

The amount of bead-immobilized prodrug 6 corresponded to a nominal concentration of 1 µM. Devices were operated for periods of 4 h in forward and reverse at ±20 nA (potential traces shown in Supplementary Fig. S14-16) followed by removal of the pumps. All wells were then incubated under static conditions for 72 h, followed by cell viability determination (Fig. 4c). Active delivery of 1 at +20 nA released CA4 from beads, which resulted in a significant reduction of cell viability to 24%, corresponding to the maximum toxic effect of CA4. In comparison, the reverse operation at -20 nA had no significant effect on cell viability, nor did operating devices at +20 nA without beads or the presence of beads with bound prodrugs without device operation.

Iontronic Release of Immobilized BSA
To verify that iontronic click-to-release can be applied to payload delivery independently of molecular weight, we demonstrated release of immobilized BSA (66 kDa) using the identical bead-based model system (Fig. 5a). Therefore, a bicyclo[6.1.0]non-4-yne (BCN) moiety was installed analogously to the synthesis of 6, which enabled the covalent attachment of commercial mono-functionalized BSA-azide (BSA-N3) to afford biotin-cTCO-BSA (7) via strain-promoted azide-alkyne cycloaddition (SPAAC). The mono-functionalization of BSA ensured site-specific conjugation and a defined 1:1 stoichiometry between protein and cTCO. The beads were subsequently coated with 7. Iontronic devices were immersed in PBS-buffered bead suspensions and operated for 1-4 h (potential traces shown in Supplementary Fig. S17-19), followed by removal of the pumps and incubation under static conditions for 24 h. Protein release was subsequently analyzed via SDS-PAGE, with bands visualized using InstantBlue® and quantified using ImageJ (v1.54p) software (Supplementary Information 8.2). The amount of protein released increased linearly with pumping duration, with a release rate of 0.11 µg (~1.5 pmol) BSA per hour of active delivery of 1 at +20 nA (Fig. 5b).

When delivering K⁺ (0.1 M KCl) as a control, we observed a background level of BSA release, which we attributed to passive protein dissociation or electrically induced detachment from the bead surface. This background was subtracted to isolate the iontronic click-to-release component, and the corresponding data are provided in Supplementary Information 8.3. These assay-related effects arise from the bead-based platform and are anticipated to be attenuated in future implementations using hydrogel-based depot architectures.

Methods
Device fabrication
Sections of polyimide-coated fused silica capillaries (CM Scientific, TSP025150-CFPH01AA, 25 µm inner diameter, 150 µm outer diameter), with a length of 500 mm, were attached to EFD precision tips (Nordson, 20FA TT 0.23”) by melting the plastic tips while the capillaries were inserted approximately 0.5 cm. The EFD precision tips were attached to syringes that were vertically connected to a N2 supply. The inner glass surface of the capillaries was etched by flushing with 2 M KOH for 2 h using N2 at a pressure of 2 bar. Next, the capillaries were washed with deionized water (10 minutes) and dried with N2 (10 minutes). An adhesion promoter (3-(trimethoxysilyl)propyl methacrylate, A 174, 10 wt% in toluene, Sigma Aldrich) was flushed for 5 h. The capillaries were flushed with EtOH (20 minutes) and dried with N2 (10 minutes). Finally, the capillaries were filled with the polyelectrolyte mixture. The polyelectrolyte mixture consisted of 2-acrylamido-2-methylpropane sulfonic acid sodium salt (AMPSA, Sigma Aldrich), poly(ethylene glycol) diacrylate Mn 575 (PEGDA, Sigma Aldrich), and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, Sigma Aldrich). The components were mixed in deionized water to achieve an 85:15 mol% AMPSA:PEGDA ratio with 0.1 mol% LAP photoinitiator at a total concentration of 25 wt%. Prior to photo-exposure, the capillaries filled with polyelectrolyte were cut from the plastic tips into pieces of approximately 150 mm length. They were mounted onto a plate using double-sided tape and the ends of the capillaries were sealed with tape. The capillaries were placed in a custom-made photo-exposure box (UV-A, 3 mW cm-2) and the CEM was polymerized for 22 h. The capillaries were further cleaved into pieces of ~15 mm with a fiber cleaver system (Fujikura CT-02) and stored in 0.1 M KCl.

Iontronic device operation
Constant-bias mode: Iontronic devices were assembled by inserting the filled 15 mm capillaries into custom 3D-printed reservoirs and mounting them onto Eppendorf tubes (0.5 mL) or 96-well plates containing a target reservoir of 40-100 µL 1× PBS (Sigma Aldrich). The source reservoir was filled with 5 mM 1 in water. To operate the IPs, electrodes (Ag/AgCl) were placed in both target and source electrolytes. The IPs were operated at constant current using an OctoStat30 (Ivium Technologies). To load the IPs with 1, the IPs were operated at +20 nA for 16-24 h. Complete loading of the IPs was indicated by a plateau in voltage (Supplementary Fig. S5).

Step-function mode: The IPs were assembled in the same manner as previously described, utilizing a 3D-printed holder that was adapted to accommodate a larger volume of target reservoir of 150 µL. The input signal was alternated periodically through levels of +20 nA (“ON”) for 16 h and zero current (“OFF”) for 7 h. A short reverse bias step (1 h, -20 nA) was incorporated between the ON and OFF steps to facilitate the retention of 1 within the channel and prevent passive diffusion in the OFF state.

Iontronic delivery of tetrazine 1
Delivery rates of 1 (in pmol min-1) were determined by operating iontronic devices at constant current (e.g., 0, +10, +20, -20 nA for set periods, typically 2-16 h). The concentration of delivered 1 in the target solution was measured using a fluorogenic assay via the C2R of 4-methylumbelliferone (4MU) from rTCO-DMEDA-4MU (2). Fluorescence measurements were performed on a plate reader (Synergy H1 microplate reader, BioTek) at 25 °C. The rTCO-DMEDA-4MU (2) probe was prepared as a 500 µM stock solution in DMSO. Tetrazine samples collected post-IP operation were diluted 1:1 into 20×PBS (8 µL sample + 8 µL 20×PBS) to reach a final buffer concentration of 10×PBS. The C2R reaction was initiated by adding 4 µL of the rTCO-DMEDA-4MU (2) stock solution to the diluted Tz samples in a 384-well microplate. The fluorescence intensity (λex = 360 nm, λem = 480 nm) was recorded over a period of 4 h at 25 °C. Tetrazine concentrations were determined from external calibration curves generated reacting 2 (100 µM) with a series of known Tz 1 concentrations (5, 10, 50, 100 µM). The fluorescence intensities after 4 h were used to determine tetrazine concentrations. The amount of delivered Tz 1 and charge as depicted in Fig. 2c was calculated as follows:
𝐷𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑡𝑒𝑡𝑟𝑎𝑧𝑖𝑛𝑒 𝟏 [𝑛𝑚𝑜𝑙] = 𝑐𝑜𝑛𝑐.𝟏 [𝑚𝑜𝑙⋅𝐿−1]⋅𝑡𝑎𝑟𝑔𝑒𝑡 𝑣𝑜𝑙𝑢𝑚𝑒 [𝐿]⋅109 (1)
𝐶ℎ𝑎𝑟𝑔𝑒 [𝑚𝐶] = 𝑐𝑢𝑟𝑟𝑒𝑛𝑡 [𝑛𝐴]⋅𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑦 𝑡𝑖𝑚𝑒 [𝑠]⋅ 10−6 (2)
Delivery rates in Fig. 2e were calculated using the following equation:
𝐷𝑒𝑙𝑖𝑣𝑒𝑟𝑦 𝑟𝑎𝑡𝑒 𝑜𝑓 𝟏 [𝑝𝑚𝑜𝑙 𝑚𝑖𝑛−1] = 𝑐𝑜𝑛𝑐.𝟏 [𝑚𝑜𝑙⋅𝐿−1]⋅𝑡𝑎𝑟𝑔𝑒𝑡 𝑣𝑜𝑙𝑢𝑚𝑒 [𝐿]⋅60 [ 𝑠 ] / 𝑚𝑖𝑛 ⋅1012 / 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑦 𝑡𝑖𝑚𝑒 [𝑠] (3)
The electrochemical efficiency was calculated by relating the experimentally delivered amount of Tz 1 to the theoretical amount of 1 defined by the total charge delivered using the following equation:
𝐸𝑙𝑒𝑐𝑡𝑟𝑜𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = 𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑇𝑧 𝟏 (𝑚𝑜𝑙) / (𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑐ℎ𝑎𝑟𝑔𝑒 (𝐶) / 2⋅𝐹) ⋅100% (4)
Here, F denotes Faraday’s constant (96 485 C mol-¹), and a charge number of 2 is used to account for the double charge of Tz 1.

Preparation of CA4 beads
Dynabeads MyOneTM Streptavidin C1 (10 mg/mL, Thermo Fisher Scientific) were used for conjugation. A 100 µL aliquot of the bead suspension was washed three times with phosphate-buffered saline (PBS) using a DynaMagTM-2 magnetic rack (Thermo Fischer Scientific) for separation. The washed beads were resuspended in 1 mL of PBS containing 5% (v/v) DMSO. To this suspension, 1 µL of biotin-cTCO-DMEDA-CA4 (6) (9 mM in DMSO) was added. The concentration of the cTCO stock solution was determined by absorbance titration using 3,6-bis(2-pyridyl)tetrazine (2Pyr2, Sigma-Aldrich), as described in Supplementary Information 3.1. Conjugation was performed at room temperature for 1 h under continuous gentle agitation. Following incubation, the CA4-functionalized beads were sequentially washed: twice with 1 mL of 5% DMSO in PBS, three times with PBS, and twice with Eagle’s minimum essential medium (E-MEM, Sigma Aldrich) containing 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mM L-glutamine, and 1% MEM Non-Essential Amino Acids. Beads were finally resuspended in the same supplemented E-MEM to a final concentration of 1 mg/mL for use in iontronic CA4 release experiments.

Preparation of BSA beads
Mono-functionalized azide-modified bovine serum albumin (BSA-N₃, 0.1 mg/mL in PBS, 200 µL, Vector Laboratories) was reacted with biotin-cTCO-BCN (S3) (10 µL, 87 mM in DMSO; 300-fold molar excess) under static conditions for 1 h at room temperature. Prior addition of the TCO, the concentration of the cTCO stock solution was determined by absorbance titration with 2Pyr2, following Supplementary Information 3.1. The reaction mixture was purified using ZebaTM Spin Desalting Columns (0.5 mL, Thermo Fisher Scientific) to remove unreacted S3. If residual S3 was detected by HPLC-MS, the purification step was repeated. For bead conjugation, 1 mL of Dynabeads MyOneTM Streptavidin C1 (10 mg/mL, Thermo Fisher Scientific) was used per 100 µL of biotin-cTCO-BSA (7) solution. Owing to the reduced binding capacity of biotinylated proteins on Dynabeads MyOneTM Streptavidin C1 beads, the quantity of beads was increased 10-fold relative to those used in the CA4-release experiments. Beads were washed three times with PBS using magnetic separation (DynaMagTM-2), then resuspended in 800 µL PBS. Subsequently, 200 µL of the BSA-conjugate 7 solution was added. The mixture was incubated for 1 h at room temperature under continuous gentle agitation. Following incubation, the BSA-bead complexes were washed five times with 1 mL PBS and resuspended in PBS to a final bead concentration of 10 mg/mL for use in iontronic BSA release experiments.

Cell culture and assays
The human GBM cell line U-87 (provided by the MUG Cell Bank, catalogue no. 300367, CLS) was cultured at 37 °C and 5% CO2 in Eagle’s minimum essential medium (E-MEM) containing 10% fetal bovine serum (FBS), 4.5 g/L glucose, 2 mM L-glutamine, and 1% MEM Non-Essential Amino Acids. All assays were performed using flat-bottomed, transparent 96-well plates (Corning). For iontronic release studies using CA4-beads, transparent HTS transwell 96-well plates with a pore size of 0.4 µm (Corning) were used. For cell proliferation studies, U-87 cells (6000 cells/well) were treated for 72 h with freshly prepared compound solutions: sulfo-cTCO-DMEDA-CA4 (4, 0.01-1000 nM), CA4 (0.01-1000 nM), 4 (0.01-1000 nM) + 1 (5 µM), 1 (5 µM). For iontronic release studies, penicillin/streptomycin was added to the media to a final concentration of 1% v/v. Cell viability was determined by MTS assay (CellTiter 96® Aqueous One Solution Cell Proliferation Assay Promega, Germany) according to the manufacturer’s protocol and was performed on a CLARIOstar plate reader (BMG Labtech, Germany) at 490 nm with top optics and orbital averaging. IC50 values were obtained by fitting data points to a sigmoidal curve using Prism 10 (GraphPad software, USA) and the following equation, in which top and bottom refer to the minimally and maximally inhibited response, respectively:
𝑌 = 𝐵𝑜𝑡𝑡𝑜𝑚+ (𝑇𝑜𝑝−𝐵𝑜𝑡𝑡𝑜𝑚) / (1+(𝐼𝐶50/𝑋)𝐻𝑖𝑙𝑙𝑆𝑙𝑜𝑝𝑒) (5)

Iontronic release of CA4 from sulfo-cTCO-DMEDA-CA4 (4) in cellular environment
Tetrazine 1 (5 mM) was loaded into the source reservoir of the iontronic devices (AMPSA/PEGDA 85/15 mol% ratio at 25 wt% in deionized water, 15 mm length, 25 µm inner diameter, 150 µm outer diameter, Ag/AgCl-source electrode). Devices were operated at constant current using an 8-channel OctoStat30 (Ivium Technologies). The channel loading was done at +20 nA over 15 h. Agar bridge electrodes were prepared by adding agar (4 wt% in 0.1 mM KCl) into custom 3D-printed funnels and covering the agar with aqueous KCl (0.1 mM). Above the agar, a freshly etched Ag/AgCl electrode was placed. Agar bridge electrodes were sterilized by 30 min UV irradiation in a laminar flow hood. 6000 U-87 cells were seeded in 96-well plates and incubated in 250 µL cell media containing 100 nM sulfo-cTCO-DMEDA-CA4 (4). Iontronic devices were installed in the cell wells, together with an agar bridge Ag/AgCl. Devices were operated at +20 nA for active delivery, or at -20 nA for reverse delivery for 1 h each. Afterwards, devices and agar bridges were removed, and plates were incubated at 37 °C and 5% CO2 and cell viability was read out using a MTS assay after 72 h.

Iontronic release of CA4 from beads
Iontronic devices (AMPSA/PEGDA 85/15 mol% ratio at 25 wt% in deionized water, 15 mm length, 25 µm inner diameter, 150 µm outer diameter, Pt-source electrode) were set up the same way as for the iontronic release of CA4 from sulfo-cTCO-DMEDA-CA4 (4) on cells (source reservoir concentration: 5 mM 1, agar bridge electrodes, 4 wt% in 0.1 mM KCl; Ag/AgCl). U-87 cells (6000 cells/well) were seeded in the transwell 96-well receiver plates and incubated in 150 µL cell media. Transwell insert plates containing 50 µL of CA4-bead suspension per well were submerged and devices installed subsequently. The remaining wells were closed with custom-made lids to prevent contamination and evaporation. Devices were operated at +20 nA for active delivery or at -20 nA for reverse delivery for 4 h each and were removed after operation. Subsequently, plates were incubated at 37 °C and 5% CO2 for 72 h and cell viability was determined as described above.

Iontronic release of BSA from beads
Iontronic devices (AMPSA/PEGDA 85/15 mol% ratio at 25 wt% in deionized water, 15 mm length, 25 µm inner diameter, 150 µm outer diameter; source reservoir concentration: 5 mM 1) were installed on 0.5 mL Eppendorf tubes using the custom 3D-printed housings. Pt-electrodes were integrated into the device design (Fig. 2b). The channel loading was done at +20 nA over 13.6 h. After reaching a steady potential indicating a fully occupied CEM, iontronic devices were installed on 0.5 mL Eppendorf tubes containing 50 µL BSA-bead suspension and run at +20 nA for different time periods (1 h, 2 h and 4 h). Delivery of K+ (0.1 M KCl in the source reservoir) was performed for 4 h instead of 1 to isolate the contribution from passive or electrochemically induced detachment of BSA from the beads. Similarly, beads were incubated for 4 h without any iontronic intervention. After 24 h following active delivery, the magnetic beads were separated from the supernatant using the DynaMagTM -2 magnetic rack and all BSA-containing samples were stored at -20 °C. For SDS-PAGE analysis, 10 µL of BSA-containing samples were mixed with 3.33 µL of Bio-Rad 4× Laemmli sample buffer (Bio-Rad, #1610747) and heated to 90 °C for 10 min. Samples were then loaded onto precast NovexTM WedgeWellTM 12% Tris-Glycine gels (Thermo Fisher Scientific, XP00120BOX) and electrophoresed at 200 V for 45 min using a Bio-Rad PowerPacTM HC system. Gels were stained with InstantBlueTM Coomassie protein stain (Abcam, ab119211) for 1 h without destaining. Imaging was performed using a Bio-Rad ChemiDocTM imaging system. Band intensities were quantified using ImageJ software (v1.54p), and protein concentrations were determined by comparison to a BSA calibration curve (0.05-1.0 µg) run on the same gels.

Statistical Analysis
Statistical analysis was performed using commercially available software (Prism 10, GraphPad, USA) and specific statistical tests were used as stated in the figure description.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Abstract\nI believe the abstract gives a clear and honest overview of the study. In my view, it already covers everything needed. The\nonly line that feels slightly overstated is the claim about “next-level electroceutical devices” — I would suggest toning that\ndown a bit.\nIntroduction\nCrucially, the introduction then introduces the novel solution in a logical way. The authors explain that by integrating\niontronic delivery with bioorthogonal click-to-release (C2R) chemistry, they decouple the transport mechanism from the\npayload activation. In my view this is a clear statement of novelty. The text and the schematic in Fig. 1 together make it easy\nto understand that instead of pumping the drug itself, they pump a chemical trigger (a charged tetrazine) which then reacts\nwith an immobilized prodrug to release the actual drug.\nIf I were to suggest any additions, it would be to perhaps cite or acknowledge any closely related prior attempts (if any) at\nremotely triggered drug release using chemical reactions. The authors have cited bioorthogonal prodrug release studies, but\nit might further strengthen novelty to explicitly contrast their approach with other stimulus-responsive drug delivery systems.\nFor example, mentioning how iontronic click-to-release differs from light-triggered or magnetically triggered release systems\n(in terms of precision or invasiveness) could be beneficial. But, this is a minor point.\nProgrammable tetrazine delivery\nI believe the correlation between charge and delivered tetrazine is pretty convincing to me, but the electrochemical efficiency\nis not fully addressed (I may have missed that part). It would strengthen this section if the authors reported at least an\napproximate charge-to-molecule conversion ratio. Without that information, it is difficult to assess how much of the applied\ncurrent contributes to actual ion transport versus leakage. But I understand these may not have been part of the study’s\ndesign. Even so, the strong linearity between applied charge and delivered amount already supports the claim of precise\nelectrical control. Adding a short statement acknowledging this limitation would make the section feel complete and\ntransparent.\nThe linear charge–delivery trend is convincing, yet the error spread in Figure 2c appears uneven and is not discussed.\nAuthors may consider specifying n and providing R² to clarify how consistent the linear response is across devices.\nCA4 Prodrug Activation\nThis section gives us good chemical evidence through HPLC that the CA4 prodrug can be efficiently cleaved in solution.\nHowever, it seems to me this analysis reflects bulk reaction conditions (please correct me if I am mistaken), while the later\ncell-based results, for example Figure 3c, only infer successful release indirectly through cytotoxicity. As a personal\nquestion, don`t authors think that, without chemical verification, it remains not more than an assumption that the same\nrelease mechanism occurs in the iontronic setup? Including or referencing a chemical confirmation of CA4 formation under\niontronic operation would bridge these two pieces of evidence and make the mechanistic link between electrical input and\nmolecular release more realistic?\nCA4 Release from Beads (Figure 4)\nHere authors performed an experiment that uses a fixed 4-hour stimulation period at ±20 nA. This may show On/off\nfunctionality but the degree of dose programmability kind of feels uncertain. However, it would help if the authors explained\nwhether shorter activation times or lower currents were tested and yielded some certain effects.\nIn Figure 5b, we can see a background release of BSA and this is shown in red in the figure. Authors wrote:\n“When KCl solution was pumped instead of tetrazine, a substantial release of BSA was still detected, which we attribute to\nan electrochemically induced bead detachment effect.”\nCould you please expand this discussion a bit. I think this part is critical.\nOther than the specific points discussed above, the figures are clear, well organized, and visually engaging; the authors\nmanaged to make a complex process easy to follow. The work demonstrates high novelty by integrating iontronic delivery\nwith click-to-release chemistry. Addressing the controlled release of both small and macromolecules was important. The\ndataset is rich. The manuscript can be accepted after addressing aforementioned minor points.", "reviewer_2": "The manuscript co-lead by Waldherr, Mikula, and Bintinger demonstrates proof-of-concept for ionotronic click-to-release\nchemistry of prodrugs and proteins. I enjoyed reading this manuscript, which spans the fields of electroceuticals,\nbioorthogonal and prodrug chemistry. The manuscript clearly describes precise spatiotemporal control using a model\nprodrug and masked protein (BSA), thus, will be of great interest to the chemical biology and medicinal chemistry fields. It is\nalso pitched to a level that is accessible for other scientists, especially those in the electrochemical and materials space.\nThe methods are described in detail with high quality experimental data that supports the conclusions of the manuscript, i.e.,\nto demonstrate spatiotemporal control of a bioorthogonal click-to-release reaction, and this manuscript will provoke interest\nin the area.\nThe proof-of-concept was demonstrated via prodrug (CA-4) activation or protein unmasking using well-established tetrazine-\nTCO click-to-release chemistry, which itself is not novel, but is novel and exciting when combined with electrochemically\ndriven spatiotemporal prodrug activation. The setup enables a controlled and pulsatile release of chemical activator\n(tetrazine) from a cleverly designed reservoir which then reacts with a TCO-prodrug (or protein) that leads to the tautomeric\ndiazine and release steps. The tetrazine is held in the reservoir that has an ion exchange membrane trapping the cationic\ntetrazine activator molecule on one side, and on the other side, payload (prodrug or enzyme) immobilised on streptavidin\nbeads (via a biotin attached to the TCO-payload). The current is applied, and the membrane is then permeable to the\ntetrazine. Reversal of the current was used to demonstrate no permeability of the tetrazine, proving that the ionotronic pump\nand an applied current is essential to achieve payload activation.\nCell viability of the prodrug alone, and immobilised on beads, showed comparable results, paving the way for similar\nsystems that are more bio-relevant than the beads used in the proof-of-concept (e.g., hydrogels). Ultimately, this approach\nwill evolve into a system that could be used in vivo (mentioned in the manuscript conclusions) and is the first step to\ntherapeutic implants that can adapt to changes in real-time, being switched off and back on again through application of an\nelectrical current.\nSomething that is not considered here is the lack of regeneration of the depot’s payload. Essentially, the TCO is consumed\nwith each reaction and eventually would be exhausted from the depot, rendering it ineffective to further tetrazine released\nfrom the ionotronic pump. However, this is another challenge, and it will be exciting to see the next generation of this system\nand its application to in vivo implants and prodrug activation strategies.\nThe following points should be considered by the authors during any revision:\nThe NMR spectra and reported data are good and of very high purity, but there are some NMR assignments that should be\ncorrected (details below).\n-Compound 2 has substantially more 13C peaks assigned than is expected. Are these due to rotamers? Also, in the spectra\nfor Compound 2, there are 7 carbon peaks in the 130-185 ppm region (page 28, ESI) but from the structure, I would expect 6\ncarbon peaks (4 x carbonyl and 2 x alkene).\n-Compound S3 has 44 assigned carbon peaks, but only 42 expected. Looking at the spectra it appears there are 42 peaks\n(page 30), so the authors must have assigned extra peaks.\n-Compound 2 and S3 have carbon assignments that are identical (e.g., 70.6, 70.6, 70.3 x 3, etc.). These peaks that are\nidentical should be assigned to 2 decimal places, to distinguish them from each other.\n-It is difficult to see using the spectra provided, but some of the coupling constants (J values) for compounds are very large\nand may not be assigned correctly. Compound 2 is an example where J values of 33 Hz and 22 Hz are reported. Are these\ncorrect? Should these peaks that are very complex be assigned as multiplets (m)?", "reviewer_3": "Claim: The authors demonstrated an iontronic system that deliver charged tetrazines as molecular scissors to selectively\nclick trans-cyclooctane linkers and release tethered molecules on demand. They showed that electronically tunable\ntetrazine delivery sustained over several days and precise release of payloads ranging from small molecules to large\nproteins. By integrating biorthogonal click-to-release chemistry with iontronic, the current work overcomes molecular size,\ncharge and stability limitations.\nStrengths of the manuscript:\n(i) Click-to-release is an interesting concept that beautifully utilizes charged tetrazines as molecular scissors to cleave the\nbond.\n(ii) This concept overcomes the limitation of delivering large molecular weight payload.\n(iii) The molecular conjugates were thoughtfully designed and characterized. The experimental demonstrations are clear in\nmost cases.\nWeaknesses of the manuscript\n(i) The concept is solely dependent on molecular design. The author has shown only one transporter molecule i.e. tetrazine.\n(ii) Solubility of the conjugate molecule might be a major concern.\nComments: Accepted after major revision required.\n1. The release kinetics of the payload are likely governed by the formulation of the cation-exchange membrane\n(PEGDA/AMPSA), since crosslink density and membrane thickness strongly influence diffusion and how long tetrazine is\nretained in the matrix. Did the authors evaluate these effects (PEGDA:AMPSA ratio, degree of polymerization/crosslinking,\nand film thickness)?\nA plot showing component concentration (or PEGDA:AMPSA ratio / crosslink density) versus tetrazine retention time (with\nmembrane thickness as a separate series or secondary axis) may clarify how membrane composition can control release\nkinetics.\n2. The rationale for using beads is not clearly explained in the manuscript. Could the authors clarify the choice. Does the\nsize of the beads influence the outcome? I guess it will.\n3. In figure 2e, for a constant current of +5 nA, the delivery rate was 0.7 ± 0.6 pmol min⁻¹. This value exhibits a very large\nrelative error. Comment on whether the applied current was too insignificant to trigger any mobility.\n4. The second-order rate constant (46 M⁻¹s⁻¹) was determined using a model cTCO compound. How well do you expect this\nkinetic value to translate to the actual prodrug (compound 4) in the cellular environment, where diffusion and steric factors\ndiffer?\n5. Did you examine the stability of the prodrug in cell culture medium or serum over time (without Tz), to confirm no slow,\nnon-specific release of CA4 occurs?\n6. The covalent attachment of biotin-cTCO to BSA via SPAAC is clear. However, it would be valuable to report the average\nnumber of cTCO groups per BSA molecule (degree of functionalization) and additionally any potential protein denaturation\nduring SPAAC attachment or immobilization could affect release and activity should be addressed."}, "author_response": "Point-by-Point Response to Reviewer Comments\nIontronic Click-to-Release Enables Electrically Controlled Delivery of Drugs and Biomolecules\nBeyond Charge and Size Limita@ons\nsubmi&ed to Nature Communica2ons\nWe thank the reviewers for their careful evalua3on and construc3ve feedback. We have revised the\nmanuscript to address all comments. Below, we provide a detailed, point-by-point response indica3ng\nthe changes made to the text. Reviewer comments are shown in italics, followed by our responses.\nReviewer #1\nGeneral Comment:\n… the figures are clear, well organized, and visually engaging; the authors managed to make a complex\nprocess easy to follow. The work demonstrates high novelty by integra@ng iontronic delivery with click-\nto-release chemistry. Addressing the controlled release of both small and macromolecules was\nimportant. The dataset is rich. The manuscript can be accepted aCer addressing aforemen@oned minor\npoints.\nResponse\nWe appreciate Reviewer #1’s detailed and posi3ve assessment of our work and their helpful\nsugges3ons for improving clarity and completeness. We have carefully addressed each of the specific\npoints raised and revised the manuscript accordingly.\nComment #1\nI believe the abstract gives a clear and honest overview of the study. In my view, it already covers\neverything needed. The only line that feels slightly overstated is the claim about “next-level\nelectroceu@cal devices” — I would suggest toning that down a bit.\nResponse\nWe have revised the phrase in the Abstract to “advanced programmable electroceu3cal devices”.\nComment #2\nIf I were to suggest any addi@ons, it would be to perhaps cite or acknowledge any closely related prior\naHempts (if any) at remotely triggered drug release using chemical reac@ons. The authors have cited\nbioorthogonal prodrug release studies, but it might further strengthen novelty to explicitly contrast\ntheir approach with other s@mulus-responsive drug delivery systems. For example, men@oning how\niontronic click-to-release differs from light-triggered or magne@cally triggered release systems\n(in terms of precision or invasiveness) could be beneficial. But, this is a minor point.\nResponse:\nWe performed two edits in the Introduc@on. First, we adapted and expanded the paragraph on\nimplantable systems to include magne3c, thermal, and ultrasound-ac3vated devices, emphasizing their\nlimited spa3al precision, poten3al invasiveness, and lack of repeatable dose control:\nImplantable devices and osmotic pumps have been successfully designed to deliver drugs locally 4,13-20,\nbut these technologies typically rely on pre-programmed release or external triggers. Such triggers can\nbe chemical (e.g., pH changes/hydrolysis21,22, enzyme mediated cleavage23,24, oxidative stress/ROS25-27)\nor physical (e.g., magnetism28-30, temperature31, ultrasound32,33 or light34-36). The latter enable remote\nactivation, converting externally applied energy into localized effects such as heating, mechanical stress,\nor photon-induced activation at the target site. Depending on the trigger platform used, this may require\nimplantable release reservoirs or local administration of stimulus-responsive carriers (e.g.,\nnanoparticles) that can be subsequently activated in situ. As activation relies on energy propagation\nthrough biological tissue, achieving precise spatial targeting and accurate dose control remains\nchallenging. In addition, such energy transfer can adversely affect surrounding tissue and, for light-\nbased systems, typically requires optical access to the target region. Iontronic pumps (IP) offer an\nalternative to several of these limitations by converting electrical signals directly into molecular\ntransport. providing tunable delivery rates with high spatiotemporal control.\nSecond, we refined the paragraph introducing click-to-release chemistry to dis3nguish between\nsystemically delivered ac3vators and the localized, electronically controlled approach presented here,\nand improved the transi3on toward the selected IEDDA reac3on:\nThis concept has previously been applied for targeted drug activation in vivo, including in hydrogel\ndepot systems and antibody-drug conjugates.51-54 Although these approaches offer localized therapeutic\naction, they depend on systemic dosing of prodrugs and afford only limited control over the timing of\nactivation. In contrast, the approach presented here demonstrates electronically controlled activation and\nenables tunable dosing. To implement this concept, we employed the click-triggered bond-cleavage\nchemistry of trans-cyclooctenes (TCO) and tetrazines (Tz), known for its exceptional kinetics and\nproven compatibility with biological systems.55\nComment #3\nI believe the correla@on between charge and delivered tetrazine is preHy convincing to me, but the\nelectrochemical efficiency is not fully addressed (I may have missed that part). It would strengthen this\nsec@on if the authors reported at least an approximate charge-to-molecule conversion ra@o. Without\nthat informa@on, it is difficult to assess how much of the applied current contributes to actual ion\ntransport versus leakage. But I understand these may not have been part of the study’s design. Even so,\nthe strong linearity between applied charge and delivered amount already supports the claim of precise\nelectrical control. Adding a short statement acknowledging this limita@on would make the sec@on feel\ncomplete and transparent.\nResponse:\nWe revised a statement in the Results sec3on quan3fying the electrochemical transport efficiency to\naddress the reviewer’s comment.\nAn increase in applied charge (current × time) could be directly correlated to the amount of delivered 1,\ndemonstrating full control over the iontronic transport (Error! Reference source not found.c) with an\nelectrochemical transport efficiency of approximately 68% at +20 nA.\nTo further clarify the calcula3on, we added the following descrip3on to the Methods sec3on:\nThe electrochemical efficiency was calculated by rela3ng the experimentally delivered amount of Tz 1\nto the theore3cal amount of 1 defined by the total charge delivered using the following equa3on:\n𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑇𝑧 𝟏 (𝑚𝑜𝑙)\n𝐸𝑙𝑒𝑐𝑡𝑟𝑜𝑐ℎ𝑒𝑚𝑖𝑐𝑎𝑙 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = ⋅100%\n𝑑𝑒𝑙𝑖𝑣𝑒𝑟𝑒𝑑 𝑐ℎ𝑎𝑟𝑔𝑒 (𝐶)\n8 = \n2⋅𝐹\nWhere F is Faraday’s constant (96 485 C mol-1) and the charge number 2 is taken into account to account\nfor the double charge of Tz 1.\nComment #4:\nThe linear charge–delivery trend is convincing, yet the error spread in Figure 2c appears uneven\nand is not discussed. Authors may consider specifying n and providing R² to clarify how consistent\nthe linear response is across devices.\nResponse:\nWe thank the reviewer for this observa3on. The number of devices (n = 4-7) and the coefficient of\ndetermina3on (R² = 0.9861) have been added to Fig. 2c to demonstrate the consistency of the linear\nresponse. The varia3on in error magnitude across charge values is characteris3c of iontronic devices\nfor this “new geometry” proof-of-principle level of development. Error levels observed at intermediate\ncharge regimes represent typical device-to-device variability, whereas the compara3vely lower\ndevia3ons at minimal and maximal charge transfer likely reflect favorable measurement condi3ons\nrather than reduced variability. A systema3c inves3ga3on of the contribu3ng factors is currently\nongoing as part of pla_orm development, but falls outside the scope of the present study, which\nprimarily aims to demonstrate proof-of-principle control rather than device op3miza3on.\nComment #5\nThis sec@on gives us good chemical evidence through HPLC that the CA4 prodrug can be efficiently\ncleaved in solu@on. However, it seems to me this analysis reflects bulk reac@on condi@ons (please\ncorrect me if I am mistaken), while the later cell-based results, for example Figure 3c, only infer\nsuccessful release indirectly through cytotoxicity. As a personal ques@on, don’t authors think that,\nwithout chemical verifica@on, it remains not more than an assump@on that the same release\nmechanism occurs in the iontronic setup? Including or referencing a chemical confirma@on of CA4\nforma@on under iontronic opera@on would bridge these two pieces of evidence and make the\nmechanis@c link between electrical input and molecular release more realis@c.\nResponse\nWe agree with the reviewer that the ini3al HPLC analysis reflected bulk reac3on condi3ons and that\ndirect verifica3on under iontronic opera3on would strengthen the mechanis3c link. To directly confirm\nthat the CA4 release mechanism occurs under iontronic opera3on, we performed addi3onal HPLC\nexperiments in which Tz 1 was delivered iontronically into a reservoir containing prodrug 4 (50 µM) for\n3, 6, and 9 h. A linear increase in CA4 forma3on was observed as a func3on of pumping dura3on. In\ncontrast, iontronic delivery of K⁺ (0.1 M KCl) for 9 h resulted in no detectable release of CA4, confirming\nboth the electrochemical stability of the prodrug and the requirement of Tz transport for ac3va3on.\nThese data have been added to the Supplementary Informa@on 6.1.\nFurthermore following sentence was added to the Results sec3on:\nTo confirm that release also occurs under iontronic conditions, we performed HPLC analysis during\nelectronic delivery of Tz 1 into a reservoir of 4. CA4 release increased proportionally with pumping\nduration (3-9 h), whereas K⁺ delivery did not induce any detectable release, confirming mechanistic\nfidelity and prodrug stability under iontronic operation (see Supplementary Information 6.1).\nThe two CA4 peaks visible in the HPLC traces correspond to the known E/Z isomers of combretasta3n\nA-4, as rou3nely observed in analy3cal separa3ons and reported in the literature (e.g., Karatoprak, G.\nŞ., Küpeli Akkol (2020). Combretasta3ns: An Overview of Structure, Probable Mechanisms of Ac3on\nand Poten3al Applica3ons. Molecules, 25(11), 2560.), hnps://doi.org/10.3390/molecules25112560.\nThis is an expected characteris3c of the drug and not indica3ve of degrada3on.\nComment #6\nHere authors performed an experiment that uses a fixed 4-hour s@mula@on period at ±20 nA. This may\nshow on/off func@onality but the degree of dose programmability kind of feels uncertain. However, it\nwould help if the authors explained whether shorter ac@va@on @mes or lower currents were tested and\nyielded some certain effects.\nResponse\nWe thank the reviewer for this comment. As detailed in our response to Comment #5, we performed\naddi3onal iontronic delivery experiments demonstra3ng that CA4 release scales propor3onally with\npumping dura3on (3-9 h), confirming dose programmability under iontronic control. This data have\nbeen included as men3oned in Supplementary Informa3on 6.1 and the Results sec3on.\nWe would like to emphasize that because CA4 is highly potent, even short s3mula3on periods deliver\nbiologically relevant doses, as stated in the manuscript: “Based on the established delivery rate of 1 at\n+20 nA, we es@mated that ac@ve pumping for just 30 seconds would suffice to deliver ~10 nM of Tz into\n250 µL, thereby releasing a biologically relevant amount of CA4.” For this reason, the experiment was\ndesigned to demonstrate safe and reversible on/off control rather than fine dosing, which is addressed\nanaly3cally and mechanis3cally in the new HPLC data.\nComment #7\nIn Figure 5b, we can see a background release of BSA and this is shown in red in the figure. Authors\nwrote: “When KCl solu@on was pumped instead of tetrazine, a substan@al release of BSA was s@ll\ndetected, which we aHribute to an electrochemically induced bead detachment effect.” Could you\nplease expand this discussion a bit. I think this part is cri@cal.\nResponse\nWe thank the reviewer for this valuable comment. To clarify the origin of the background signal, we\nemphasize that the click-cleavable linker is chemically stable under iontronic condi3ons. As\ndemonstrated in the addi3onal HPLC experiment provided in response to Comment #5 (Supplementary\nFig. S10), pumping K⁺ does not induce any release from prodrug 4. Because the BSA conjugate employs\nthe same cTCO linker architecture, we infer equivalent chemical stability in this system.\nGiven the absence of chemically triggered release under K⁺ pumping, we anribute the background\nobserved in the BSA experiment to non-specific effects associated with the bead-based assay format,\ne.g. passive protein dissocia3on or electrically induced detachment from the bead surface. To present\nthe iontronically triggered component more clearly and avoid possible misinterpreta3on, we corrected\nthe data in Fig. 5b in the Results sec3on for background due to non-specific release, while the\nunderlying raw data was added to the Supplementary Informa3on in Sec3on 8.3 for full transparency.\nThis clarifica3on does not alter the conclusions of the study. It ensures that the electronically triggered\nrelease resul3ng from tetrazine delivery is accurately represented. In subsequent itera3ons of the\npla_orm, we have begun to explore hydrogel-based depot architectures, in which such non-specific\neffects are an3cipated to be anenuated.\nAccordingly, the following sentence was revised in the Results sec3on to bener reflect this explana3on:\nWhen delivering K⁺ (0.1 M KCl) as a control, we observed a background level of BSA release, which\nwe attributed to passive protein dissociation or electrically induced detachment from the bead surface.\nThis background was subtracted to isolate the iontronic click-to-release component, and the\ncorresponding data are provided in Supplementary Information 8.3. These assay-related effects arise\nfrom the bead-based platform and are anticipated to be attenuated in future implementations using\nhydrogel-based depot architectures.\nReviewer #2\nGeneral Comment\nThe manuscript co-lead by Waldherr, Mikula, and Bin@nger demonstrates proof-of-concept for\nionotronic click-to-release chemistry of prodrugs and proteins. I enjoyed reading this manuscript, which\nspans the fields of electroceu@cals, bioorthogonal and prodrug chemistry. The manuscript clearly\ndescribes precise spa@otemporal control using a model prodrug and masked protein (BSA), thus, will be\nof great interest to the chemical biology and medicinal chemistry fields. It is also pitched to a level that\nis accessible for other scien@sts, especially those in the electrochemical and materials space.\nResponse\nWe thank Reviewer #2 for their posi3ve assessment and apprecia3on of our interdisciplinary approach\ncombining iontronics and bioorthogonal chemistry. We are pleased that the clarity and accessibility of\nthe manuscript were well received.\nComment #1\nSomething that is not considered here is the lack of regenera@on of the depot’s payload. Essen@ally,\nthe TCO is consumed with each reac@on and eventually would be exhausted from the depot, rendering\nit ineffec@ve to further tetrazine released from the ionotronic pump. However, this is another challenge,\nand it will be exci@ng to see the next genera@on of this system and its applica@on to in vivo implants\nand prodrug ac@va@on strategies.\nResponse\nWe agree with the reviewer that the current system is designed for single-use ac3va3on of immobilized\npayloads. The present work was intended as a proof-of-concept demonstra3on, using the bead system\nas a simplified model pla_orm to establish electronically controlled click-to-release chemistry and to\nconfirm precise, programmable ac3va3on of both small and macromolecular payloads.\nIn the context of highly potent compounds such as CA4, regenera3on becomes less cri3cal, since\nrealis3c depot architectures could serve as effec3vely inexhaus3ble reservoirs. Based on feasibility\nes3ma3ons, drug-polymer constructs with approximately 1% (w/w) loading can reach up to 100 µM\nreleasable drug concentra3ons, several orders of magnitude above typical IC₅₀ values for cytotoxins (1–\n200 nM). Such capaci3es would enable many ac3va3on cycles without significant deple3on, even\nwithout re-func3onaliza3on of the reac3ve sites. From preliminary in vivo studies, administering a\nsustained local chemotherapy dosing at the tumor site to flank tumors in mice, we observed that the\ndrug dosing can be reduced to only 5% of the systemically efficient dose, s3ll reaching efficient tumor\ncontrol.\nThis concept, however, extends beyond the scope of the present study, which focused on establishing\nfeasibility and mechanism using the bead system as a model for future hydrogel-based depots.\nComment #2\nThe NMR spectra and reported data are good and of very high purity, but there are some NMR\nassignments that should be corrected (details below).\n• Compound 2 has substan@ally more 13C peaks assigned than is expected. Are these due to\nrotamers? Also, in the spectra for Compound 2, there are 7 carbon peaks in the 130–185 ppm\nregion (page 28, ESI) but from the structure, I would expect 6 carbon peaks (4 × carbonyl and 2\n× alkene).\n• Compound S3 has 44 assigned carbon peaks, but only 42 expected. Looking at the spectra it\nappears there are 42 peaks (page 30), so the authors must have assigned extra peaks.\n• Compound 2 and S3 have carbon assignments that are iden@cal (e.g., 70.6, 70.6, 70.3 × 3, etc.).\nThese peaks that are iden@cal should be assigned to 2 decimal places, to dis@nguish them from\neach other.\n• It is difficult to see using the spectra provided, but some of the coupling constants (J values) for\ncompounds are very large and may not be assigned correctly. Compound 2 is an example where\nJ values of 33 Hz and 22 Hz are reported. Are these correct? Should these peaks that are very\ncomplex be assigned as mul@plets (m)?\nResponse\nWe thank the reviewer for the careful inspec3on of the NMR data. The following correc3ons and\nclarifica3ons were implemented in the Suppor@ng Informa@on:\n• For Compound 2, we confirmed that the apparent excess 13C peaks result from rotameric\nconforma3ons, which produce duplicate carbonyl signals. This observa3on is consistent with\nprevious reports for similar carbamate-containing structures, where Compound 2 has been\nreported as a mixture of rotamers. The following explanatory sentence was added to the\ncap3on: Compound 2 is reported as a mixture of rotamers and iden3cal peaks were assigned 2\ndecimal places to dis3nguish the signals. The 13C-NMR code reads now:\n13C NMR (151 MHz, CD Cl ) δ 160.73, 160.70, 156.0, 155.9, 155.59, 155.57, 154.56, 154.53, 154.49,\n2 2\n154.4, 154.32, 154.29, 154.1, 154.0, 153.9, 152.57, 152.56, 132.1, 132.0, 132.0, 131.9, 125.68, 125.65,\n125.62, 125.56, 118.7 118.52, 118.45, 118.4, 117.8, 117.7, 117.6, 114.5, 114.42, 114.40, 114.37, 110.7,\n110.5, 110.4, 75.0, 74.9, 74.8, 74.7, 48.0, 47.8, 47.5, 47.4, 47.3, 47.2, 46.7, 46.5, 41.2, 41.1, 41.0, 36.39,\n36.36, 36.21, 36.18, 36.16, 35.8, 35.64, 35.62, 35.5, 35.4, 35.2, 35.0, 34.6, 29.5, 29.4, 24.8, 24.7, 24.5,\n18.9.\n• For Compound S3, two wrongly assigned carbon signals were removed, and iden3cal peaks\nwere assigned 2 decimal places to dis3nguish the signals. The 13C-NMR code reads now:\n13C NMR (151 MHz, CD Cl ) δ 180.7, 173.2, 163.7, 157.1, 156.1, 132.0, 131.8, 99.1, 72.5, 70.8, 70.8, 70.6,\n2 2\n70.5, 70.40, 70.38, 70.2, 70.1, 63.0, 62.1, 60.5, 55.9, 54.2, 54.0, 53.8, 53.7, 53.5, 46.1, 44.6, 41.3, 41.2,\n41.0, 39.7, 39.6, 36.30, 36.29, 36.1, 31.6, 31.4, 29.5, 28.5, 28.4, 26.0, 21.7, 20.5, 18.2, 18.1.\n• For Compound 2, overly large J values were re-evaluated. The relevant resonances are indeed\ncomplex mul3plets, and their coupling constants have been updated or reported as mul3plets\n(m) where appropriate. The 1H-NMR code reads now:\n1H NMR (600 MHz, CD Cl ) δ 7.59 (ddd, J = 11.8, 8.5, 2.8 Hz, 1H), 7.13 – 7.05 (m, 2H), 6.19 (s, 1H), 5.86 –\n2 2\n5.70 (m, 1H), 5.53 (t, J = 15.5 Hz, 1H), 3.69 – 3.56 (m, 2H), 3.55 – 3.41 (m, 3H), 3.10 (d, J = 10.7 Hz, 2H),\n3.01 (dd, J = 12.7, 6.1 Hz, 4H), 2.94 (d, J = 7.6 Hz, 2H), 2.11 – 1.88 (m, 4H), 1.83 (?, J = 14.4, 7.2 Hz, 1H),\n1.73 – 1.54 (m, 3H), 1.51 – 1.42 (m, 1H), 1.13 – 0.99 (m, 1H), 0.85 – 0.73 (m, 1H).\nReviewer #3\nGeneral Comment\nThe authors demonstrated an iontronic system that deliver charged tetrazines as molecular scissors to\nselec@vely click trans-cyclooctane linkers and release tethered molecules on demand. They showed that\nelectronically tunable tetrazine delivery sustained over several days and precise release of payloads\nranging from small molecules to large proteins. By integra@ng biorthogonal click-to-release chemistry\nwith iontronic, the current work overcomes molecular size, charge and stability limita@ons.\nStrengths of the manuscript:\n(i) Click-to-release is an interes@ng concept that beau@fully u@lizes charged tetrazines as molecular\nscissors to cleave the bond.\n(ii) This concept overcomes the limita@on of delivering large molecular weight payload.\n(iii) The molecular conjugates were thoughrully designed and characterized. The experimental\ndemonstra@ons are clear in most cases.\nResponse:\nWe thank Reviewer #3 for the posi3ve and encouraging evalua3on. We appreciate the recogni3on of\nthe novelty and clarity of our approach, as well as the acknowledgment of the system’s ability to\nachieve electronically controlled, on-demand release of both small molecules and proteins. The\ncomments accurately capture the conceptual advance of coupling iontronic delivery with\nbioorthogonal click-to-release chemistry, and we are pleased that the strength and design of the\nmolecular conjugates were well received.\nWeaknesses of the manuscript (i/ii)\nComment (i)\nThe concept is solely dependent on molecular design. The author has shown only one transporter\nmolecule, i.e., tetrazine.\nResponse\nThe focus of the present study was not to establish a general mechanism of iontronic transport, which\nhas already been demonstrated in the literature for a variety of drug molecules already men3oned in\nthe Introduc3on of the current paper, but rather to show that click-to-release chemistry can be\nintegrated with iontronic delivery to enable remotely controlled ac3va3on. We therefore selected one\nrepresenta3ve charged tetrazine species that allows precise electrical addressing and mechanis3c\nclarity. This approach ensured that the study could clearly demonstrate the feasibility of electrically\ntriggered bioorthogonal release. We fully agree that extending this principle to other click-to-release\ntriggers will further broaden the chemical scope of this ac3va3on strategy, and this is the direc3on of\nour current work. While not shown in this manuscript, our preliminary experiments with addi3onal\ncharged tetrazine triggers, including benzylic amine 4-(6-methyl-1,2,4,5-tetrazin-3-\nyl)phenyl)methanamine, as well as the unsymmetrical variant of 1 - 2-(6-methyl-1,2,4,5-tetrazin-3-\nyl)ethan-1-amine (see. Sarris, Chem. Eur. J. 2018, 24, 18075,\nhnps://doi.org/10.1002/chem.201803839), indicate comparable iontronic transport and click-to-\nrelease performance. This supports that the concept is not limited to a single tetrazine structure, and\na systema3c evalua3on of this broader tetrazine library is currently ongoing.\nComment (ii)\nSolubility of the conjugate molecule might be a major concern.\nResponse\nWe appreciate this point and confirm that all compounds used in this study were designed to exhibit\nadequate aqueous solubility under the experimental condi3ons. The Tz 1 carries two permanent\ncharges, ensuring good solubility and efficient iontronic transport. The sulfonated CA4 conjugate 4 and\nits released product CA4 were proven to be soluble at the concentra3ons applied in both chemical and\nbiological assays (see, e.g., Keppel, ChemBioChem 2022, 23, e202200363.\nhnps://doi.org/10.1002/cbic.202200363), and BSA is known to remain fully soluble and structurally\nstable under similar buffered condi3ons (see, e.g., Peters, Adv. Protein Chem. 1985, 37, 161–245.\nhnps://doi.org/10.1016/S0065-3233(08)60065-0).\nWe also note that solubility is not expected to be a limi3ng factor in future advanced depot structures,\nsuch as hydrogels, where the conjugates are immobilized within a solid support. In such systems, only\nthe solubility of the released payloads will be relevant. Here the electrical control over release rate\nallows precise adjustment to prevent local precipita3on even for less soluble payloads.\nComment #1\nThe release kine@cs of the payload are likely governed by the formula@on of the ca@on-exchange\nmembrane (PEGDA/AMPSA), since crosslink density and membrane thickness strongly influence\ndiffusion and how long tetrazine is retained in the matrix. Did the authors evaluate these effects\n(PEGDA:AMPSA ra@o, degree of polymeriza@on/crosslinking, and film thickness)?\nA plot showing component concentra@on (or PEGDA:AMPSA ra@o / crosslink density) versus tetrazine\nreten@on @me (with membrane thickness as a separate series or secondary axis) may clarify how\nmembrane composi@on can control release kine@cs.\nResponse\nWe fully agree that the membrane composi3on and thickness cri3cally affect ion transport and\nreten3on. In the present work, our goal was to demonstrate the feasibility and electronic control of\ntetrazine delivery, and therefore we employed a standardized PEGDA/AMPSA formula3on op3mized in\nprevious iontronic studies to ensure reproducible transport characteris3cs. For previous studies see:\nCorrelaFng Ionic ConducFvity and Microstructure in Polyelectrolyte Hydrogels for Bioelectronic\nDevices - Macromol. Rapid Commun (2022).\nhnps://doi.org/10.1002/marc.202100687\nTranslaFng Electronic Currents to Precise Acetylcholine-Induced Neuronal Signaling using an Organic\nElectrophoreFc Delivery Device - Advanced Materials (2009).\nhnps://doi.org/10.1002/adma.200900187\nCapillary-fiber based electrophoreFc delivery device - ACS Applied Materials & Interfaces (2019).\nhnps://doi.org/10.1021/acsami.8b22680\nA decade of iontronic delivery devices - Advanced Materials Technologies (2018).\nhnps://doi.org/10.1002/admt.201700360\nImpact of PEGMA on transport and co-transport of methanol and acetate in PEGDA-AMPS caFon\nexchange membranes - Journal of Membrane Science (2022).\nhnps://doi.org/10.1016/j.memsci.2021.119950\nTo avoid introducing addi3onal variables that would obscure the rela3onship between applied charge\nand tetrazine dose, we deliberately kept PEGDA:AMPSA ra3o, crosslinking condi3ons, and membrane\nthickness constant.\nA systema3c inves3ga3on of how membrane composi3on, crosslinking degree, and thickness influence\nrelease kine3cs is indeed important and represents the next step toward applica3on-specific\nop3miza3on. However, this extends beyond the scope of the current proof-of-concept study, which\nfocused on coupling iontronic delivery with chemical ac3va3on.\nComment #2\nThe ra@onale for using beads is not clearly explained in the manuscript. Could the authors clarify the\nchoice? Does the size of the beads influence the outcome?\nResponse\nStreptavidin-coated magne3c beads were selected as a well-defined, commercially available model\npla_orm that allows highly reproducible immobiliza3on of bio3n-tagged conjugates via a well-\nestablished bio3n-streptavidin interac3on. Their surface-based presenta3on of cTCO-func3onalised\nmolecules enables precise control over the number of reac3ve sites and facilitates quan3ta3ve release\nanalysis. While bead size may, in principle, affect total surface area, all experiments were carried out\nusing the same batch of beads under iden3cal condi3ons, ensuring internal comparability.\nThe bead setup was therefore intended as a rapid and analy3cally accessible proof-of-concept system\nto demonstrate iontronic control over chemical release. It does not represent a final depot design for\nin vivo use. Ongoing work focuses on advancing the immobiliza3on strategy toward hydrogel-based\ndepot architectures, which are more suitable for biological environments and scalable therapeu3c\nimplementa3on.\nTo clarify this in the manuscript, we updated the sentence in the Results sec3on as follows:\nBuilding on these results, we employed cTCO-bis-NHS (5) to synthesize biotin-cTCO-DMEDA-CA4\n(6), enabling immobilization of the prodrug onto streptavidin-coated magnetic beads, which were\nselected as a well-defined solid support allowing quantitative analysis of release while serving as a\nsimplified proof-of-principle model system.\nComment #3\nIn Figure 2e, for a constant current of +5 nA, the delivery rate was 0.7 ± 0.6 pmol min⁻¹. This value\nexhibits a very large rela@ve error. Comment on whether the applied current was too insignificant to\ntrigger any mobility.\nResponse\nWe would like to clarify that iontronic transport was clearly observed at +5 nA, confirming that the\napplied current was sufficient to drive tetrazine delivery. The larger rela3ve error at this low current\nstems from the inherent variability of the first-genera3on devices, as indicated by the comparable\nabsolute standard devia3on across all current levels. While the rela3ve varia3on appears larger at +5\nnA due to the smaller delivered amount, the underlying transport mechanism remains fully ac3ve and\nscales linearly with the applied charge.\nWe are currently working on further op3miza3on of both the device architecture and analy3cal assays,\naiming to improve precision and reproducibility in future genera3ons of iontronic delivery systems. The\noverall linear correla3on between applied charge and delivered amount remains consistent, confirming\nrobust electrical control even in this ini3al prototype configura3on.\nComment #4\nThe second-order rate constant (46 M-¹.s-¹) was determined using a model cTCO compound. How well\ndo you expect this kine@c value to translate to the actual prodrug (compound 4) in the cellular\nenvironment, where diffusion and steric factors differ?\nResponse\nWe agree that the cellular environment can influence the apparent click-to-release kine3cs. To address\nthis directly, we determined the kine3cs in a representa3ve cell-culture medium (Fluorobrite\nsupplemented with 10% FBS), which yielded a second-order rate constant of 37 M-¹.s-¹, confirming that\nthe reac3on proceeds efficiently under physiologically relevant condi3ons.\nThe experiment was added to the Suppor@ng Informa@on (Supplementary Fig. S1) and the second-\norder rate constant was adapted in the Results sec3on:\nDetermining rate constants with a model TCO is a standard prac3ce to benchmark intrinsic reac3vity\nfor related conjugates that share the same reac3ve core - e.g.:\nUnraveling Tetrazine-Triggered Bioorthogonal EliminaFon Enables Chemical Tools for Ultrafast\nRelease and Universal Cleavage - Journal of the American Chemical Society (2018).\nhnps://doi.org/10.1021/jacs.7b11217\nThe overall click-to-release process is governed primarily by the used Tz-TCO combina3ons with limited\ninfluence from the nature of the leaving group/payload, suppor3ng the use of model systems for rate\nbenchmarking.\nBeyond the current study, we are developing next genera3on click-to-release linkers based on the\nrecently reported iTCO scaffold, which shixs control of the post click cascade to the TCO and enables\nultrafast, near quan3ta3ve release across a broad range of tetrazines and biological media. Integra3ng\niTCO into our iontronic pla_orm is expected to further increase release efficiency under physiologically\nrelevant condi3ons, but this lies beyond the scope of the present work.\nPre-print: hnps://chemrxiv.org/engage/chemrxiv/ar3cle-details/67ce0a786dde43c90833b680\nComment #5\nDid you examine the stability of the prodrug in cell culture medium or serum over @me (without Tz),\nto confirm no slow, non-specific release of CA4 occurs?\nResponse\nThe stability of the iden3cal prodrug (compound 12, sulfo-cTCO-DMEDA-CA4) has been\ncomprehensively characterized in our previous work (ChemBioChem, 2022 – Reference 42 in the\noriginal manuscript). HPLC analysis confirmed that the compound remained fully stable in both PBS\nand cell growth medium (DMEM + 10 % FBS) at 37 °C for 120 h, showing no detectable release of CA4.\nOnly par3al trans-to-cis isomeriza3on of the cTCO linker (≈ 33 % axer 72 h, ≈ 44 % axer 120 h) was\nobserved, while the DMEDA bis(carbamate) linkage remained intact, confirming the integrity of the\nprodrug under physiological condi3ons.\nThese findings verify that the CA4-TCO prodrug 4 used in this study is chemically stable and that drug\nrelease occurs exclusively upon tetrazine ac3va3on. Cell-viability data for 4 in Fig. 3b in the current\nmanuscript further confirms this assump3on.\nPlease note that, such trans to cis isomeriza3on would render the click-to-release pathway ineffec3ve\nfor that frac3on of molecules, but it would merely keep CA4 covalently caged rather than generate free\ndrug. From a safety perspec3ve, this means that no unintended payload is released and that\nisomeriza3on can only reduce the frac3on of prodrug that remains addressable, not create background\ntoxicity.\nTetrazine-Triggered Bioorthogonal Cleavage of trans-Cyclooctene-Caged Phenols Using a Minimal\nSelf-ImmolaFve Linker - ChemBioChem (2022). hnps://doi.org/10.1002/cbic.202200363\nComment #6\nThe covalent aHachment of bio@n-cTCO to BSA via SPAAC is clear. However, it would be valuable to\nreport the average number of cTCO groups per BSA molecule (degree of func@onaliza@on), and\naddi@onally any poten@al protein denatura@on during SPAAC aHachment or immobiliza@on could affect\nrelease and ac@vity should be addressed.\nResponse\nThe BSA–azide used in this study is a commercial mono-func3onalized deriva3ve (modified at the single\naccessible cysteine residue), which ensures a defined 1:1 correla3on between the reac3ve site and the\nreleased protein. We added the following clarifica3on in the manuscript:\nTo verify that iontronic click-to-release can be applied to payload delivery independent of molecular\nweight, we demonstrated release of immobilized BSA (66 kDa) using the identical bead-based model\nsystem (Error! Reference source not found.a). Therefore, a bicyclo[6.1.0]non-4-yne (BCN) moiety\nwas installed analogously to the synthesis of 6, which enabled the covalent attachment of commercial\nmono-functionalized BSA-azide (BSA-N ) to afford biotin-cTCO-BSA (7) via strain-promoted azide-\n3\nalkyne cycloaddition (SPAAC). The mono-functionalization of BSA ensured site-specific conjugation\nand a defined 1:1 stoichiometry between protein and cTCO. Regarding denatura3on: SPAAC is widely\napplied under mild, aqueous condi3ons for protein bioconjuga3on (including in vivo labeling), and\nstreptavidin-bio3n magne3c bead systems-specifically Dynabeads™ MyOne™ Streptavidin C1 are\nrou3nely used for protein capture/immobiliza3on in immunoassays and pull-downs under non-\ndenaturing condi3ons. Based on this established prac3ce and manufacturer guidance, we do not\nexpect denatura3on under our experimental condi3ons – see:\nDynabeads™ MyOne™ Streptavidin C1 – product protocol page (Thermo Fisher):\nhnps://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isola3on-and-\nanalysis/protein-isola3on-protocol/dynabeads-myone-streptavidin-c1.html\nExample of protein immobilizaFon on streptavidin surfaces under non-denaturing assay condiFons\n(supports common use in ELISA/pull-downs): PLOS ONE (2018): “Bio3n-tagged proteins: Reagents for\nefficient ELISA-based an3body analysis”\nhnps://journals.plos.org/plosone/ar3cle?id=10.1371%2Fjournal.pone.019131"}, {"round": 2, "reviewer_comments": {"reviewer_1": "In my view, the authors have adequately addressed all of the concerns I raised, and I believe the manuscript is suitable for\npublication without the need for further revisions.", "reviewer_2": "As in my initial review, this is an excellent manuscript reporting on proof-of-concept for iontronic click-to-release chemistry of\nprodrugs and proteins. The authors have provided a detailed response to all reviewer comments. In my opinion, they have\naddressed the comments well and have made the necessary adjustments to the manuscript, including the characterization\ndata requested by this reviewer.", "reviewer_3": "I am happy with the revised manuscript as the authors have successfully addressed the concerns."}, "author_response": ""}]

Decision letter