A click chemistry-based, free radical-initiated delivery system for the capture and release of payloads

Abstract

Click chemistries are efficient and selective reactions that have been leveraged for multi-stage drug delivery. A multi-stage system allows independent delivery of targeting molecules and drug payloads, but targeting first-phase materials specifically to disease sites remains a challenge. Stimuli-responsive systems are an emerging strategy where common pathophysiological triggers are used to target payloads. Oxidative stress is widely implicated in disease, and we have previously demonstrated that reactive oxygen species (ROS) can crosslink and immobilize polyethylene glycol diacrylate (PEGDA) in tissue mimics. To build on these promising results, we present a two-step, catch-and-release system using azide-DBCO click chemistry and demonstrate the capture and eventual release of a fluorescent payload at defined times after the formation of a PEGDA capturing net. The azide component is included with radical-sensitive PEGDA, and the payload is conjugated to the DBCO group. In cell-free and cell-based tissue mimic models, azides were incorporated at 0–30% in the first-phase polymer net, and DBCO was delivered at 2.5–10 µM in the second phase to control payload delivery. The payload could be captured at multiple timepoints after initial net formation, yielding a flexible and versatile targeting system. Matrix metalloproteinase (MMP)-degradable peptides were incorporated into the polymer backbone to engineer fluorescent payload release by MMPs, which are broadly upregulated in diseases, through degradation of the capture net and directly from the DBCO. Taken together, this research demonstrates proof-of-principle for a responsive and clickable biomaterial to serve as a multi-potent agent for the treatment of diseases compounded by high free radicals.

Keywords:

• Drug delivery
• click chemistry
• stimuli-responsive
• release
• PEGDA

Introduction

Targeted drug delivery is an established strategy to increase the concentration of a therapeutic in a tissue of interest. The overarching goal is to limit the exposure of healthy tissues to the therapy and curb unintended side effects (Zhao et al., 2020). In traditional targeting systems, the drug and targeting moiety are concurrently administered, which inherently links the biodistribution of the therapeutic and the targeting agent (Parker et al., 2019). Click chemistry reactions have emerged as a strategy for multi-stage, targeted delivery (Hapuarachchige et al., 2016; Hapuarachchige, 2017). The reactions are named for their high specificity and yield, and select click pairs are bioorthogonal, biocompatible, and can proceed in living systems (Kim & Koo, 2019; Takayama et al., 2019). Azide and dibenzocyclooctyne (DBCO) are a bioorthogonal click pair that have been leveraged for drug delivery. Disease tissues are pretargeted with the azide component, and the drug is delivered in a second phase, coupled with DBCO (Hapuarachchige et al., 2016; Takayama et al., 2019). A two-phase approach allows the properties of each phase to be independently optimized and enhances the flexibility of delivery timing and component combinations (Hapuarachchige, 2017).

The success of click chemistry-based delivery depends on the specificity of pretargeting, ensuring that only cells in the diseased tissue are tagged with azides. Current approaches rely on established techniques, such as antibody-ligand binding or local injections, to deliver first-phase materials (Hapuarachchige et al., 2016; Takayama et al., 2019). For antibody-ligand-mediated delivery, molecules upregulated on diseased cells are identified, and click-containing molecules are linked to antibodies that bind these overexpressed receptors (Tewabe et al., 2021). This approach is limited by the variability in receptor presentation between patients and across different disease types, but stimuli-responsive targeting can address this issue (Rosenblum et al., 2018).

Stimuli-responsive targeting leverages intra- or extracellular factors that are dysregulated in many diseases, rather than relying on disease-specific molecules. Enzyme expression, pH, redox potential, and oxygen levels are broad triggers for delivery, and targeting physiological factors enables the development of a platform system (Thomas et al., 2020; Majumder & Minko, 2021). Free radicals, such as reactive oxygen species (ROS), are highly reactive species with an unpaired electron and are commonly overexpressed in diseased tissues (Pham-Huy et al., 2008). In a previous study, we demonstrated that ROS can crosslink and immobilize polyethylene glycol diacrylate (PEGDA), a biocompatible polymer, within tissue mimics, and that these polymers can carry and thereby deliver fluorescent payloads (Lowe et al., 2019).

In this research, we build on our stimuli-responsive, polymeric system to develop a two-phase targeting technology that leverages click chemistry reactions. ROS-sensitive PEGDA and acrylate-PEG-azide are delivered in the first phase, pretargeting tissue mimics with elevated free radical levels. The payload, tethered to DBCO, is subsequently introduced in the second phase, and capture is achieved through the azide-DBCO reaction (Figure 1). Our central aim is to establish proof-of-concept for this two-phase system, demonstrating the advantages and flexibility conferred by combining a stimuli-responsive platform system and two-phase delivery.

Figure 1. Summary of the multi-phase targeting system. Overview of the two-phase, catch-and-release targeting approach including click chemistry capture and MMP-initiated release.

Once accumulated at the disease site, drugs must be released from the polymer net to have a therapeutic effect (Wang et al., 2016). We incorporate enzymatically-sensitive peptides into first- and second-phase materials to engineer payload release. Matrix metalloproteinase (MMP) enzymes, such as collagenases, are responsible for extracellular matrix homeostasis and are broadly upregulated in disease (Löffek et al., 2011; Serra, 2020). Peptide sequences cleaved by MMPs are commercially available and can be incorporated into polymer backbones for both release and long-term network degradation and clearance. The first-phase polymer net will target and capture payloads through click chemistry, and enzymatically degradable sequences will allow local, sustained release.

Materials

Ten-thousand Dalton PEGDA (HO009009-10K) and 10,000 Dalton acrylate-PEG-azide (HE009006-10K) were purchased from Biopharma PEG Scientific. Cy5-DBCO (A130) and DBCO-Sulfo-NHS Ester (A124) were purchased from Click Chemistry Tools. Two-thousand Dalton PEGDA (ACRL-PEG-ACRL-2000) was purchased from Laysan Bio. The cystine-flanked VPM peptide sequence (LT255422) was purchased from LifeTein. Agarose (A0576), 6000 Dalton PEGDA (701963), horseradish peroxidase (HRP—P8375), acetylacetone (P7754), hydrogen peroxide (H₂O₂—216763), HEPES (H3537), sodium hydroxide (NaOH—S2770), penicillin/streptomycin (P4458), Trypsin (T4049), 10× MEM (M0275), and phosphate-buffered saline (PBS—P5493) were purchased from Sigma-Aldrich. DMEM (11885092) was purchased from Thermo Fisher Scientific. Fetal bovine serum (FBS—FBS001) was purchased from Fisher Scientific. Rhodamine B-PEG-acrylate (2000 Daltons) was custom ordered from Creative PEGWorks. Amicon^® Ultra-15 Centrifugal Filter Units (3 KDa MWCO—UFC900308; 10 KDa MWCO–UFC901024) and Roche Collagenase/Dispase^® enzyme were purchased from Millipore Sigma. A porous polyethylene (PPE) sheet (136380518) was purchased from Small Parts. Irgacure 2959 was the generous gift of the BASF Corporation. Type-I bovine collagen (C857) was purchased from Elastin Products Company.

Methods

Formation of agar microbeads

Agarose beads were prepared by dissolving agar powder at 2 wt% in 1× PBS. The solution was continuously stirred until boiling to allow the complete dissolution of the agar. A P20 pipette was used to quickly aspirate and expel 10 µL of hot agar solution, forming a droplet at the end of the pipette. The agar droplet was submerged in ice-cold 1× PBS for 30 s to allow gelation to proceed. Microbeads were stored at room temperature (RT) in 1× PBS for up to 4 weeks.

Two-phase click chemistry capture in agar microbeads

H₂O₂, HRP, and acetylacetone were used to generate ROS in vitro. All conditions were prepared and washes were performed with 1× PBS, pH 7.4. Due to the relatively large molecular size of the HRP enzyme, agar microbeads were incubated in HRP and 5 mM PEGDA/acrylate-PEG-azide (10,000 Dalton) for 2 h at RT under constant rocking. H₂O₂ and acetylacetone were added to initiate ROS formation, and microbeads were rocked for 3 h at RT to allow crosslinking to proceed. Agar beads were moved to a 1.8 mL wash solution and placed on a rotator (Rotator Genie, Scientific Industries, Inc.) at RT overnight. The following day, beads were transferred to a solution containing 10 µM Cy5-DBCO and rotated for 3 h, then moved to a fresh wash solution and rotated overnight to remove any non-captured fluorophore. Beads were placed in individual wells of a 96-well plate, and residual fluorescence in the agar beads was measured with a Tecan Infinite M200 Pro Plate Reader (excitation: 649 nm, emission: 680 nm). All experiments were performed in triplicate, data are reported as the mean ± SD, and data were statistically analyzed using a one-way ANOVA followed by Tukey’s post-hoc test unless otherwise stated.

Modulating ROS levels in the two-phase capture system

All components of the ROS initiation system were serially diluted to control the level of free radicals generated. The highest ROS level contained 1.5 mM H₂O₂, 0.19 mg/mL HRP, and 90 mM acetylacetone. It is critical to maintain a constant ratio of H₂O₂ to HRP to ensure that HRP activity is not inhibited by excess H₂O₂ (Danielson et al., 2018). To evaluate the impact of free radical concentration on click capture, the initial PEGDA solution was doped 10% with 5 mM acrylate-PEG-azide across all conditions. Residual bead fluorescence was read at 1 day and 4 days post-Cy5-DBCO addition, and wash supernatant was replaced with fresh 1× PBS every 24 h. As a control for hindered diffusion due to polymer net formation, a condition with 0% acrylate-PEG-azide (PEGDA only) at the highest ROS level was included.

Independent control of first-phase azide doping and second-phase payload dosage

The ROS concentration was fixed at the 1 mM H₂O₂ level for all remaining experiments. To characterize the impact of azide pretargeting on payload capture, the first-phase PEGDA solution was doped 0–30% with acrylate-PEG-azide. Following the reaction with ROS and washing, agar beads were incubated with 10 µM Cy5-DBCO, washed overnight at RT, and residual fluorescence was read after 24 h. In a separate experiment, acrylate-PEG-azide doping was fixed at 10% across conditions, and different doses of Cy5-DBCO were delivered (2.5, 5, and 10 µM). Residual agar bead fluorescence was measured after an overnight wash.

Payload delivery at multiple time points

Agar beads were pretargeted with PEGDA and 10% acrylate-PEG-azide and washed overnight to remove uncrosslinked polymers. Immediately after washing, either 10 or 0 µM Cy5-DBCO was delivered to beads for 3 h, beads were washed, and Cy5 fluorescence was measured with a plate reader. All beads were re-washed for another 2 days after the initial payload delivery, and a second round of Cy5-DBCO delivery was performed. Beads that did not receive payload (0 µM) in the first round were incubated with 10 µM Cy5-DBCO in the second round, and some beads that received 10 µM payload in the first round were administered a subsequent, second round dose. Post-wash residual fluorescence after both rounds of Cy5-DBCO delivery was measured with a plate reader.

Local payload capture within agar beads

Irgacure 2959, a photoinitiator that generates free radicals when exposed to UV light, was used to produce synthetic radicals with spatial control. First-phase polymers and 1 wt% Irgacure were allowed to penetrate agar beads in 96-well plates, and beads were exposed to UV light for 60 s on the 20× objective of an Olympus IX81 inverted fluorescence microscope using the DAPI filter. Beads were washed, incubated with Cy5-DBCO, re-washed, and spatial payload capture was measured on the 4× objective of the microscope. Brightfield and Cy5 fluorescent images were acquired and image analysis was performed in FIJI.

Synthesis of degradable PEGDA

An enzyme-cleavable VPM peptide flanked with terminal cysteines (GCRDVPMSMRGGDRCG) was purchased commercially. The peptide was reconstituted at 10 mM in 10× PBS, and 2000 Dalton PEGDA was prepared at 10 mM in deionized water. PEGDA and the VPM peptide were mixed in an amber vial at a 4:1 molar ratio and rotated for 2 h at 4 °C to allow acrylates and thiols to react via a Michael-type addition. The product was diluted 1:2 in deionized water and filtered through a 0.2 µm syringe filter. The filtrate was further purified to isolate the target product (acrylate-PEG-VPM-PEG-acrylate, ∼6000 Daltons) using centrifugal filter units with 3000 and 10,000 Dalton cutoffs. Solutions >10,000 Daltons and <3000 Daltons were discarded to remove off-target reaction products, and 3 spin cycles were performed with each centrifugal unit in deionized water. The remaining product was lyophilized until complete dryness or about 4 days.

Catch and release of fluorescent payloads via degradable PEGDA

Agar microbeads were used to evaluate the capture and subsequent enzyme-initiated release of a Cy5 payload, as previously described. Degradable PEGDA was used as the first-phase polymer, doped 10% with acrylate-PEG-azide. Six-thousand Dalton, non-degradable PEGDA was used as a molecular weight-matched control for capture. Bead fluorescence was read after Cy5-DBCO incubation and washing but before enzyme addition to measure click-mediated capture. Collagenase/Dispase enzyme was reconstituted to 1 mg/mL in 1× PBS, and beads were rocked in the enzyme solutions at 37 °C overnight. Residual bead fluorescence was measured after 24 h using a plate reader. Different ratios of degradable: non-degradable PEGDA were explored in one replicate, and a lower enzyme concentration (5 µg/mL) was used to characterize the sustained release profile. The fresh enzyme solution was added to beads every 24 h, and residual fluorescence was measured at 3-days and 7-days post-enzyme addition.

Synthesis of a degradable payload linker

An MMP-degradable peptide with only a C-terminal cysteine (GRDVPMSMRGGDRCG) was custom ordered from GenScript. Rhodamine B-PEG-acrylate was prepared at 9 mM in deionized water and mixed at a 1.5:1 molar ratio with 15 mM degradable monocysteine peptide in 10× PBS. The reaction was rocked at 4 °C for 2 h to allow acrylate-thiol coupling. DBCO-Sulfo-NHS Ester was dissolved at 22.5 mM in deionized water immediately before use, and 1 mL was added directly to the 3.5 mL reaction solution. The reaction was rocked for another 2 h at 4 °C to allow primary amine coupling. The reaction product was diluted in deionized water, pushed through a 0.2 µm syringe filter, and purified using a 3000 Dalton molecular weight cutoff centrifugal filter (3 spin cycles) against deionized water. The purified product was lyophilized for 4 days and stored under Argon at −20 °C.

Catch and release of fluorescent payloads via a cleavable linker

PEGDA doped with 30% acrylate-PEG-azide was crosslinked with ROS in agar beads, and the DBCO-VPM-PEG-Rhodamine B synthesis product was delivered. DBCO-VPM-PEG-Rhodamine B was incubated in agar beads for 8 h to allow the click chemistry reaction to proceed. Beads were washed for 3 days, and 1 mg/mL Collagenase/Dispase enzyme was added to initiate release. Residual Rhodamine B fluorescence was read pre- and post-enzyme addition with a plate reader (excitation: 540 nm, emission: 586 nm).

Click capture in cell-laden collagen gels

Human dermal fibroblasts (Life Technologies, Carlsbad, CA, USA) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Confluent fibroblasts were trypsinized, resuspended in fresh media, and mixed with buffered, type-I bovine collagen at 150,000 cells/mL. Buffered collagen was prepared according to the following recipe for 1 mL: 20 µL 50× HEPES, 86 µL 0.15 N NaOH, 100 µL 10× MEM, 117 µL concentrated cell solution, and 677 µL 3.75 mg/mL collagen. The collagen solution was plated into PPE annular rings housed in 48-well tissue culture plates. The plates were incubated at 37 °C for 1 h to allow hydrogel formation, and media was added on top of the gels. Gels were allowed to compact in the z-direction via cell-generated forces for 2 days. PEGDA, acrylate-PEG-azide, and Irgacure (photoinitiator) were added to cultures, and polymers were allowed to penetrate gels for 2 h. Gels were then exposed to UV light using the DAPI filter of a fluorescence microscope (20× objective) for 3 min to locally generate free radicals. Cell-laden gels were washed overnight in fresh media, Cy5-DBCO was delivered to gels for 4 h, and gels were re-washed in phenol red-free media for 4 days. Residual fluorescence was measured via microscopy and image analysis was performed in FIJI.

Fully-compacted, fixed, cell-laden gels to evaluate payload capture

Fibroblast-laden collagen gels were prepared as previously described, but gels were plated in polydimethylsiloxane (PDMS) annular molds (0.5in inner diameter) adhered to 12-well tissue culture plates. Once plated, collagen-cell solutions were incubated for 1 h at 37 °C to initiate gelation. PDMS molds were removed from the well plates, fresh media was added, and gels were detached from the cell culture surface. Floating gels can freely compact in all directions, and gels were incubated for 2 days before fixation in 4% paraformaldehyde. Fixed gels were incubated with PEGDA, acrylate-PEG-azide, and Irgacure, and the center of the gel was exposed to UV light. Cy5-DBCO was delivered for 4 h, washed, and fluorophore capture was visualized using microscopy.

Results

Free radical concentration determines the amount of payload captured

Agar microbeads were used as a model to evaluate free radical-initiated delivery of a Cy5 payload. PEGDA doped with 10% acrylate-PEG-azide was added to agar beads, ROS at 0–1.5 mM H₂O₂ were generated within the agar to initiate crosslinking, and washes were performed to remove unreacted species. Cy5-DBCO was delivered in the second phase, and beads were re-washed before measuring fluorescence, shown in Figure 2. Higher H₂O₂ levels resulted in significantly increased residual Cy5 fluorescence (*p < .001), and no capture was observed when ROS was absent from the beads. In conditions where acrylate-PEG-azide was delivered, there was no significant decrease in Cy5 signal from wash day 1 to 4, demonstrating that the captured payload was retained. In the no azide condition, there was a significant decrease in fluorescence between washes (**p < .001), which accounts for slower payload clearance due to polymer net formation.

Figure 2. Two-phase payload delivery depends on the ROS level. PEGDA/acrylate-PEG-azide was delivered to agar beads with different concentrations of ROS. The capture of Cy5-DBCO via click chemistry was measured with a plate reader. The level of Cy5 capture directly related to the amount of ROS (*p < .001), and the payload was sustained after 4 days of washing, except in the no azide control, where significant payload loss was observed after 4 days of washing (**p < .001). H₂O₂ levels >0.25 mM resulted in a significant (*p < .001) increase in residual fluorescence compared to 0 mM ROS and no azide negative controls.

Modular payload delivery is independently controlled

ROS levels were fixed at 1 mM H₂O₂ and doping of acrylate-PEG-azide in the first phase was varied from 0 to 30%. After Cy5-DBCO delivery and washing, the amount of Cy5 retained in agar beads was measured (Figure 3(A)). Payload capture is directly related to the amount of azide administered in the first phase, with higher amounts of acrylate-PEG-azide resulting in more capture, up to 30% doping (p < .001). Azide levels above 30% were not explored due to the saturation of the fluorophore at higher capture levels. Minimal residual Cy5 fluorescence was observed when either acrylate-PEG-azide or ROS were excluded. In a subsequent study, acrylate-PEG-azide doping was fixed at 10% in the first-phase PEGDA network and the Cy5-DBCO concentration was varied. As seen in Figure 3(B), higher initial Cy5 dosages resulted in higher residual fluorescence levels after washing (p < .001).

Figure 3. Click chemistry capture directly depends on both first-phase azide doping and second-phase payload dose. H₂O₂-derived ROS and PEGDA/acrylate-PEG-azide were delivered to agar beads to generate a polymer network. Beads were washed and Cy5-DBCO was delivered. Residual fluorescence was measured with a plate reader after the beads were re-washed. (A) The level of acrylate-PEG-azide doping was varied from 0 to 30% to modulate the amount of click-reactive azides that were trapped, and 10 µM Cy5-DBCO was delivered. Pre-wash fluorescence intensity is represented as a dashed line. A condition without ROS initiators was included as a negative control. Higher azide inclusion resulted in significantly more capture, and the level of fluorophore capture is directly related to the azide concentration. (B) Acrylate-PEG-azide doping was fixed at 10%, different concentrations of Cy5-DBCO were delivered, and bead fluorescence was measured pre- and post-washing. Delivering more Cy5-DBCO resulted in significantly higher capture levels, indicating that the delivered dose can be controlled independently of first-phase conditions. (*p < .001).

Pretargeting confers flexibility in drug delivery timing

The PEGDA/acrylate-PEG-azide network was formed via ROS-initiation in agar beads, and either 10 or 0 µM Cy5-DBCO was delivered immediately after washing. Beads were re-washed for 2 days, and a second round of Cy5-DBCO was delivered to select conditions (Figure 4). For the condition where the payload was only delivered immediately, fluorescence was sustained at the 2-day measurement after additional washing. When Cy5-DBCO was delivered at the later timepoint only (0 + 10 µM), residual fluorescence mirrored immediate delivery results. In the 10 + 10 µM condition (Cy5-DBCO administered in both the first and second rounds), there was a significant increase in residual fluorescence after the second round of Cy5-DBCO delivery (p < .001) and the magnitude was ∼2× the immediate delivery measurement.

Figure 4. Payload administration at multiple timepoints. Agar beads were pretargeted with azide-containing polymer nets, and two rounds of Cy5-DBCO delivery were performed. Beads that received Cy5-DBCO in the first round had a significant increase in fluorescence compared to controls (*p < .001), and the payload was sustained after washing. When Cy5-DBCO was delivered only in the second round (after additional washing of the polymer net), capture was comparable to immediate delivery results. This demonstrates that capture can be achieved after initial net formation, enhancing the flexibility of the system. When Cy5-DBCO was delivered in both dosing rounds (10 + 10 µM), the captured fluorescence nearly doubled, indicating that multiple doses can be subsequently delivered.

Spatially-resolved, two-phase capture

Free radicals were produced within a precise area of the agar beads, defined by the beam of UV light delivered through the 20× microscope objective. The border of the agar bead is visualized under brightfield imaging (Figure 5). Cy5 fluorescence is only detected within a defined cylinder of the agar. Cy5-DBCO cleared from non-UV exposed areas of the agar, as negligible fluorescent signal was detected at the bead border.

Figure 5. Fluorescent payload is captured only in the region with free radicals. Agar beads were imaged in brightfield using the 4× objective of a microscope, and the entire bead is within the image frame. Irgacure and first-phase polymers were delivered, and the center of the bead was exposed to UV light (20× objective) to generate radicals. Cy5-DBCO was delivered after washing, and beads were re-washed to remove unreacted Cy5-DBCO. Fluorescence was only detected in the center of the bead, corresponding to the region where free radicals were generated. This demonstrates that capture net formation is specific and local to areas with high levels of free radicals.

Enzymatically-degradable PEGDA allows payload release and clearance

MMP-degradable PEGDA was synthesized and delivered to agar beads. Click capture levels were comparable to non-degradable PEGDA and significantly higher than the no azide negative control (p < .001). After incubating with an exogenous collagenase enzyme for 24 h, Cy5 completely cleared beads with degradable PEGDA, but the payload was retained with non-degradable PEGDA (Figure 6). A lower enzyme concentration and different ratios of degradable to non-degradable PEGDA were used in Figure 7. Slower, sustained release over a 7-day period was achieved at the lower enzyme concentration, and manipulating the degradable polymer ratio allowed for control over the total mass of the payload that was released. No Cy5 capture was observed in the PEGDA-only (0% acrylate-PEG-azide) control.

Figure 6. Enzyme-initiated release is achieved with degradable PEGDA. An MMP-sensitive peptide sequence was incorporated into the PEGDA backbone to create an enzymatically-degradable polymer (MMP-PEGDA). MMP-PEGDA was delivered to agar beads with 10% acrylate-PEG-azide, and a non-degradable PEGDA condition was included as a positive control for capture. Both degradable and non-degradable conditions had a significant increase in fluorescence compared to the no azide control, indicating that the synthesized product was able to crosslink within the agar and capture Cy5-DBCO. Beads were incubated with a collagenase enzyme for 24 h, and the payload was fully released after 24 h in the degradable PEGDA condition. The Cy5 payload was retained in the non-degradable control. (*p < .001).

Figure 7. Payload release is controlled by the ratio of degradable to non-degradable PEGDA. Different ratios of non-degradable PEGDA and the MMP-degradable polymer-peptide conjugate were delivered to agar beads, doped 10% with acrylate-PEG-azide. ROS initiators were added to induce crosslinking, and Cy5-DBCO was delivered. Before enzyme addition, all azide-containing conditions had comparable capture to the non-degradable control (0:100). To initiate release, 5 µg/mL collagenase was added and fluorescence was read at 3 and 7 days of washing. Conditions with at least 50% MMP-degradable PEGDA had significantly less residual fluorescence than the non-degradable control (*p < .01), demonstrating that the payload can be released via enzymatic cleavage. Payload release is directly related to the amount of MMP-degradable PEGDA included in the initial capture net.

Selective payload release using an enzymatically-cleavable linker

Rhodamine B was coupled to DBCO via an MMP-degradable peptide (VPM), and non-degradable PEGDA/acrylate-PEG-azide nets within agar beads were used to evaluate click capture potential. As shown in Figure 8, conditions with both components of the capture net (PEGDA and acrylate-PEG-azide) had a significantly higher residual fluorescence compared to PEGDA-only and acrylate-PEG-azide–only negative controls (p < .001). After the addition of the collagenase, residual fluorescence in the capture net condition was significantly reduced (p < .001) and on par with negative controls. When no enzyme was added, the fluorescence was retained in the beads.

Figure 8. Catch and release of a target payload via enzymatic degradation. PEGDA doped with 30% acrylate-PEG-azide was incubated with agar beads, and ROS was generated to crosslink capture nets within the beads. DBCO–MMP-degradable (VPM)–Rhodamine B was subsequently delivered and captured, indicated by a significant increase in residual fluorescence compared to PEGDA-only and azide-only controls. Rhodamine B was released from beads upon enzyme addition, evidenced by a significant drop in fluorescence after the 24 h incubation. Fluorescence was sustained when no enzyme was added. (*p < .001).

Two-phase payload capture in a mock tissue system

Human fibroblasts were seeded in collagen gels within PPE rings to create a 3D tissue model. After cells compacted the gels, first-phase polymers and Irgacure were delivered, and UV light was used to locally generate radicals in the center of each gel. Significantly higher (p < .001) Cy5 levels were observed in the condition where both initial polymers were delivered and free radicals were generated (Figure 9). In a subsequent experiment, cell-laden gels were allowed to freely compact without a fixed boundary condition to create a denser tissue. Compacted gels were fixed to facilitate analysis. Microscopy was used to visualize capture, and there is a distinct region in the center of the gel with a brighter residual fluorescence, which corresponds to the UV-exposed region (Figure 10).

Figure 9. Payload captured via click chemistry in cell-laden collagen gels. Collagen gels with human fibroblasts were generated in PPE annular rings, allowing compaction in the z-direction and the formation of a tissue-like structure. PEGDA, acrylate-PEG-azide, and Irgacure (IRG) were introduced to cultures, and the center of each gel was exposed to UV light. Cy5-DBCO was delivered and residual fluorescence was quantified after 4 days of washing (left). Representative images of each condition are shown (right). When capture net polymers and IRG were administered and UV-exposed (E), significantly higher fluorescence (*p < .001) was observed in the UV-region compared to controls (A–D).

Figure 10. Multi-stage click capture in densely-compacted, fixed gels. Collagen gels were detached from culture surfaces, allowing gels to float in the media and freely compact via cell-generated forces before fixation. Fixed gels were incubated with PEGDA, acrylate-PEG-azide, and Irgacure, exposed to UV light in the inner region of the gel, and Cy5-DBCO was delivered. Fluorescence imaging was used to visualize click capture. Cy5-DBCO shows high background in fixed cells, so the entire gel can be seen in the image. There is a distinct region in the center of the gel with a brighter residual fluorescence, which corresponds to UV-exposed region. This indicates that polymers can crosslink within the dense, cell-laden gel to form a polymer net which can subsequently capture a clickable payload.

Discussion

We have demonstrated that acrylated PEG polymers can pretarget both cell-free and cellular tissue models with high levels of exogenously produced free radicals. Either ROS or UV-exposed Irgacure initiates polymer crosslinking, generating polymer nets that immobilize the coupled azide molecule. A DBCO-linked fluorescent payload administered in a second phase is captured via click chemistry reaction. Peptides that are enzymatically cleaved can be incorporated into both first- and second-phase material, and enzyme exposure triggers payload release. This catch-and-release system can target and concentrate payloads to areas with excess free radicals, and payloads can be locally released at the disease site via MMPs.

Agarose is a biostructural molecule that can readily be formed into beads for use as a cell-free tissue phantom for high-throughput studies. We generated free radicals through HRP-catalyzed reduction of H₂O₂ using 0–1.5 mM H₂O₂ and an acetylacetone mediator (Danielson et al., 2018). Although reported H₂O₂ concentrations at diseased sites are elevated relative to healthy tissues, in vivo concentrations remain in the micromolar range. We used a single bolus of free radicals at a higher concentration to model the continuous, lower radical levels in vivo (Gupta et al., 2012; Weinstain et al., 2014; Zhang et al., 2019). We found that the amount of payload captured within agar was directly dependent on the H₂O₂ concentration. This indicates that when more free radicals are present, increased crosslinking of the initial polymer network occurs, leading to a higher amount of payload captured. This is critical, as we expect that higher ROS levels at the disease site will initiate crosslinking and capture, whereas lower levels in healthy tissues will minimally couple the polymers and allow them to clear.

The amount of fluorophore captured could be controlled at multiple levels due to the modular nature of a two-phase system. Both acrylate-PEG-azide doping and Cy5-DBCO concentration could independently determine the amount of payload captured. Polymer networks sufficiently crosslinked in agar, even with up to 30% inclusion of a monoacrylated azide, and higher doping levels may be possible to maximize capture. The payload could be delivered either immediately after net formation or at a later timepoint, and an additional payload could be captured at that later timepoint. These results highlight the flexibility of a multi-phase system, and the potential to deliver different therapeutics or diagnostics with a single capture net. As currently formulated, we are not saturating all available azides in the pretargeted net. Although we established this system with the azide-DBCO reaction, other click pairs with faster kinetics, such as tetrazine and trans-cyclooctene (TCO), are becoming widely used and may promote more efficient capture (Kim & Koo, 2019).

Payload release is a critical component of delivery, as the payload that remains attached to the polymer network is unable to act as a soluble molecule. We demonstrated that we can incorporate an enzymatically-sensitive peptide sequence into both the PEGDA backbone and the payload linker. Including this peptide sequence allows the fluorophore to be released from the immobilized polymer net upon enzyme exposure. We manipulated the ratio of degradable to non-degradable PEGDA, which allowed for control over the amount of payload released and slower release kinetics. While degradable PEGDAs can be used to modulate bulk network dynamics and long-term degradation, the released payload remains tethered to the polymer material, which will limit diffusion. A degradable linker would specifically release only the payload. We only explored a single MMP-degradable peptide here, but a library of peptides with different sensitivities has been characterized and may allow us to independently tune bulk degradation and release kinetics.

We anticipate that in vivo, endogenously-derived free radicals will serve as the stimuli for polymer crosslinking. Here, we used nonpathogenic human fibroblasts in collagen gels to generate a model for dense tissue. Fibroblasts rapidly compacted the collagen into a dense tissue-mimic, but radicals generated from fibroblasts alone were not sufficient to crosslink PEG capture nets. This is expected, as fibroblasts are not a primary source of extracellular radicals in disease. We expect that a co-culture of pathogenic and immune cell types will be needed to generate the sustained source of elevated free radicals encountered in disease states. Through the fibroblast-laden collagen model, we were able to demonstrate that polymers can crosslink within a dense matrix and in the presence of cells and cell-generated molecules.

Conclusion

We have demonstrated proof-of-concept for a free radical-initiated and click-chemistry mediated drug delivery system. PEGDA doped with acrylate-PEG-azide was immobilized in agar tissue mimics upon free radical exposure, and this polymer net was capable of capturing DBCO-coupled payloads. Payload capture directly depended on the free radical concentration, and capture could be independently controlled by modulating the concentration of either acrylate-PEG-azide or Cy5-DBCO. Second-phase Cy5-DBCO could be captured multiple days after polymer net formation, and multiple rounds of payload could be captured with the same initial net. These results highlight the versatility and enhanced simplicity conferred by a two-phase system. Enzymatically-degradable peptides were incorporated into both the PEGDA backbone and the DBCO-payload linker. Bulk network degradation was achieved with degradable PEGDA, whereas the payload was selectively released using the degradable DBCO-payload. Different ratios of enzymatically-degradable and non-degradable PEGDA were used to control the amount of payload released. In cell-based tissue mimics, local crosslinking was achieved by generating radicals with a photoinitiator and controlled irradiation with UV light. Payloads were successfully captured in areas where free radicals were generated and cleared from surrounding areas. Together, these results demonstrate strong evidence for a free radical-initiated and click chemistry-mediated delivery system.

Author contributions

Conception and design: E.T.D., K.K.S., and D.I.S.; analysis and interpretation of the data: E.T.D.; drafting of the paper: E.T.D.; revising it critically for intellectual content: E.T.D., K.K.S., and D.I.S.; final approval of the version to be published: E.T.D., K.K.S., and D.I.S. All authors agree to be accountable for all aspects of the work.

Acknowledgments

The authors would like to thank Francois Berthiaume and Suneel Kumar from the Department of Biomedical Engineering at Rutgers University for providing human dermal fibroblasts for in vitro studies.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author, DIS, upon reasonable request.

Additional information

Funding

This research was funded by the Rutgers University TechAdvance Fund (FP10535), the NIH Biotechnology Training Program (NIH T32 GM008339), the New Jersey Health Foundation (PC 123-22), an NIH Institutional Research and Career Development Award (K12GM093854), and a New Jersey Commission on Cancer Research pre-doctoral fellowship (COCR22PRF002).