(T1130-05-28) ROS-Responsive Polymeric Chloroquine Nanogel as Oral Maintenance Therapy for Inflammatory Bowel Disease

Purpose: The primary goal in treating active Inflammatory Bowel Disease (IBD) is to induce remission through intensive therapy and subsequently maintain this remission long-term with treatments that are sustainable and have minimal side effects [1]. Traditional therapies often become less effective over time and can lead to systemic immune suppression and toxicity due to their non-specific actions [2-5]. This project aims to utilize targeted oral delivery to maintain remission in IBD, offering the benefits of being non-invasive, highly compliant, selectively targeting the colon, and minimizing side effects. A key characteristic of IBD is the overproduction of reactive oxygen species (ROS), which increase by 10-1000 fold at the site of inflammation [6]. In this study, we introduce a novel thioketal linker used to synthesize crosslinked polymeric chloroquine (PCQ) particles that form nanogels in water. Chloroquine (CQ) is currently being tested in clinical trials for its efficacy in treating IBD. However, long-term CQ/HCQ treatment can cause serious side effects, including retinopathy, liver toxicity, and blurred vision, due to its high level of systemic exposure [7-10]. These nanogels are designed to protect the drug as it passes through the gastrointestinal tract, disintegrate in the presence of ROS for targeted delivery at the site of inflammation, and minimize off-target toxicities.

Methods: We synthesized a thioketal linker with hydroxy ethyl methacrylate attached at both ends and confirmed its structure with ¹ H NMR. This thioketal monomer, along with chloroquine methacrylate and hydroxypropyl methacrylate monomers, were polymerized using reversible addition-fragmentation chain transfer polymerization at various feed ratios. This process yielded PCQ nanogels with four distinct particle sizes, designated as T1, T2, T3, and T4. CQ loading was measured by peak integration using ¹H NMR. We assessed the particle size and zeta potential of the nanogels using a NanoBrook Omni particle size and zeta potential analyzer. ROS responsiveness was tested by incubating nanogels in 10 mM KO₂ for 24 hours and confirming disintegration with transmission electron microscopy (TEM). The cytotoxicity of the nanogels was tested using a cell titer blue assay on mouse intestinal (IEC6) and macrophage (RAW264.7) cells. The therapeutic efficacy was observed in a murine model of IBD induced by Citrobacter rodentium (Citro). Body weight was monitored as an indicator of toxicity. Hematoxylin and eosin-stained colon sections were analyzed by a blinded pathologist and histopathological scores were assigned based on previously described criteria [11]. Additionally, crypt heights were measured to assess the extent of hyperplastic crypt formation caused by inflammation in the Citro-induced IBD model

Results: The synthesis process for the nanogels is illustrated in Figure 1a. We synthesized and characterized four sets of nanogels ranging in size from 240 nm to 680 nm, as summarized in Figure 1d. Transmission electron microscopy (TEM) images (Figure 2a, ii) showed that the TK-crosslinked PCQ nanoparticles are nearly spherical and larger on average than the non-crosslinked PCQ nanoparticles (Figure 3a, i). Exposure of all PCQ nanogel nanoparticles to KO₂ led to the cleavage of the thioketal links and subsequent disintegration of particle morphology, as depicted in Figures 2a, v and 2a, vi (T1 and T2 shown as examples). In contrast, PCQ nanoparticles without crosslinking exhibited no significant morphological changes after KO₂ incubation. Toxicity tests revealed that all PCQ nanogels were non-toxic up to 400 µM of equivalent HCQ concentrations, maintaining cell viability above 90%. However, free HCQ showed significant cytotoxicity at concentrations above 200 µM (IC50) for IEC6 cells and 33 µM (IC50) for RAW264.7 cells (Figures 2b & 2c). Continuous administration of HCQ for seven days following Citro infection significantly reduced mouse body weight, whereas those treated with nanogels did not exhibit significant weight changes (Figure 3b). The T1 and T3 nanogel formulations significantly reduced overall colonic mucosal damage, unlike HCQ alone (Figure 3d). Additionally, the T3, T4, and HCQ treatments significantly reduced hyperplastic crypt formation in the colon due to inflammation (Figure 3e).

Conclusion: The study successfully demonstrated the synthesis of ROS-responsive PCQ nanogels. These nanogels facilitated ROS-triggered dissociation of thioketal-crosslinked PCQ nanoparticles, enabling the targeted release of PCQ at sites of inflammation. PCQ is a macromolecular drug synthesized by our lab previously and has demonstrated preferential accumulation in colon as compared to HCQ [12]. In this study, the cytotoxicity assessments indicated that the PCQ nanogel formulation substantially mitigated the cytotoxic effects of HCQ and enhanced its biocompatibility. Supporting these findings, data from the therapeutic study showed that HCQ treatment led to significant weight loss in mice, whereas mice treated with PCQ nanogels did not experience comparable weight reduction, highlighting the reduced systemic toxicity of the nanogel-delivered HCQ. Notably, the T3 nanogel formulation not only matched the local therapeutic effects of HCQ in the colon but also further enhanced its overall therapeutic efficacy. This suggests that PCQ nanogels could offer a promising alternative for maintenance therapy in IBD, providing effective local action with significantly reduced systemic side effects.

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Acknowledgements: Financial support from the University of Nebraska Medical Center and the National Institutes of Health (R01 DK124095) is acknowledged. The authors declare no conflict of interest.


Figure 1. a) Scheme for PCQ nanogel synthesis. b) 1H NMR of TK-NH2 (400 MHz, CDCl3, 25 °C) (δ(ppm) 6.00(HC=C-, a), 5.47 (-HC=C-, b), 4.23 (-OCH2-, c), 2.75 (-CH2NHCO-, d), 2.53 (-CH2S-, e), 1.81 (CH3C=C-, f), 1.46 (-CH3, g)) c) 1H NMR of PCQ nanogel in d6 DMSO. d) Table summarizing the nanogels prepared and their characteristics. 1-Feed ratio of thioketal linker and chain transfer agent (CTA). 2-CQMA content determined by 1H NMR. 3-Size and Zeta Potential measured using DLS.


Figure 2. a) TEM images of PCQ and PCQ nanogels before and after incubation with 10mM KO2 for 24 h. (Magnification (164000x). b) Effect of PCQ nanogels on cell viability of IEC6 c) RAW264.7 cell lines. CTB assay was used to determine viable cells after 24 h of treatment with increasing HCQ equivalent concentrations of treatments (0,0.3,6.3,12.5,25,50,100,200,400 µM). d) Table summarizing IC50 values of PCQ nanogels and HCQ.


Figure 3. Therapeutic efficacy of PCQ nano gels in C.rodentium induced IBD mice. a) Dosing regimen and group description for therapeutic study. b) Mice weight variation over the therapeutic study. c) Representative images of H&E stained colon (20x magnification). d) Total histopathological scoring by a blinded pathologist. e) Colon crypt length in nm.