Optical Imaging Agents Research Articles

Emissively enhanced novel azo compounds featuring with ESIPT core

This study presents the synthesis and detailed characterization of novel azo compounds featuring Excited State Intramolecular Proton Transfer (ESIPT) cores derived from orcinol, resorcinol, and 2-naphthol. Using a comprehensive suite of spectroscopic techniques, including Nuclear Magnetic Resonance (NMR), Liquid Chromatography-Mass Spectrometry (LC-MS), Ultraviolet-Visible (UV–Vis) spectroscopy, and fluorescence spectroscopy, we explored the unique photophysical properties of these compounds. Notably, the synthesized compounds exhibited exceptionally large Stokes shifts, with values recorded at 204 nm, 251 nm, and 168 nm for compounds 1A, 2A, and 3A, respectively. These shifts indicate significant potential for applications in areas such as optical sensing and fluorescent imaging. Furthermore, Density Functional Theory (DFT) calculations were employed to investigate the nonlinear optical (NLO) properties of the enol and keto tautomeric forms of these compounds. The findings revealed that compound 2A, in particular, demonstrated impressive first-order hyperpolarizability, especially in chloroform, underscoring its suitability for advanced nonlinear optical applications. The study highlights the pivotal role of the ESIPT mechanism, where efficient intramolecular hydrogen bonding facilitates proton transfer upon excitation, leading to enhanced fluorescence properties. This phenomenon is particularly pronounced in compound 2A, where dual O—H interactions contribute to its remarkable fluorescence behavior, making it a standout candidate for use in high-performance optical materials. These findings not only advance our understanding of azo compounds and their versatile photophysical characteristics but also open new avenues for their application in cutting-edge technologies such as optical sensors, imaging agents, laser materials, and other fluorescence-based devices. By strategically leveraging the ESIPT process and the unique structural features of these compounds, this research paves the way for future innovations in the field of advanced optical materials and photonic devices.

Synthetic Efforts toward the Synthesis of a Fluorinated Analog of 5-Aminolevulinic Acid: Practical Synthesis of Racemic and Enantiomerically Defined 3-Fluoro-5-aminolevulinic Acid.

In 2017, the FDA authorized 5-aminolevulinic acid (5-ALA) for intraoperative optical imaging of suspected high-grade gliomas. This was the first authorized optical imaging agent for brain tumor surgery to enhance the visualization of malignant tissue. Herein we report the synthesis of a racemic and enantiopure fluorinated analog of 5-ALA, i.e., 3-fluoro-5-aminolevulinic acid (3F-5-ALA). We anticipate that these studies will provide the foundation for the future construction of a fluorine-18-labeled 5-ALA PET tracer to be used for functional and metabolic imaging of gliomas.

A Computational Framework for the Administration of 5-Aminovulinic Acid Before Glioblastoma Surgery.

5-Aminolevulinic Acid (5-ALA) is the only fluorophore approved by the FDA as an intraoperative optical imaging agent for fluorescence-guided surgery in patients with glioblastoma. The dosing regimen is based on rodent tests where a maximum signal occurs around 6h after drug administration. Here, we construct a computational framework to simulate the transport of 5-ALA through the stomach, blood, and brain, and the subsequent conversion to the fluorescent agent protoporphyrin IX at the tumor site. The framework combines compartmental models with spatially-resolved partial differential equations, enabling one to address questions regarding quantity and timing of 5-ALA administration before surgery. Numerical tests in two spatial dimensions indicate that, for tumors exceeding the detection threshold, the time to peak fluorescent concentration is 2-7h, broadly consistent with the current surgical guidelines. Moreover, the framework enables one to examine the specific effects of tumor size and location on the required dose and timing of 5-ALA administration before glioblastoma surgery.

Theranostic Phthalocyanine and Naphthalocyanine Nanoparticles for Photoacoustic Imaging and Photothermal Therapy of Tumors.

Background: Phthalocyanine (PC) and naphthalocyanine (NC) dyes have long garnered interest as theranostic agents for optical imaging and phototherapy due to their near-infrared absorbance, photostability, imaging contrast, and proven safety in clinical trials. Yet, only a small fraction of these dyes has been evaluated as photothermal therapy (PTT) agents for cancer treatment. Methods: Nearly 40 distinct NC and PC dyes were encapsulated within polymeric PEG-PCL micelles via oil-in-water emulsions. The optimal NC/PC-loaded micelle formulations for PTT and photoacoustic (PA) imaging were identified through in vivo and in vitro studies. Results: The most promising candidate, CuNC(Octa)-loaded micelles, demonstrated a strong PA signal with a peak absorbance at ~870 nm, high photothermal efficiency, and photostability. The CuNC(Octa)-loaded micelles exhibited heat generation as good or better than gold nanorods/nanoshells and >10-fold higher photoacoustic signals. Micelle preparation was reproducible/scalable, and the CuNC(Octa)-loaded micelles are highly stable under physiological conditions. The CuNC(Octa)-loaded micelles localize within tumors via enhanced permeability and retention and are readily detectable by PA imaging. In a syngeneic murine tumor model of triple-negative breast cancer, CuNC(Octa)-loaded micelles demonstrate efficient heat generation with PTT, leading to the complete eradication of tumors. Conclusions: CuNC(Octa)-loaded micelles represent a promising theranostic agent for PA imaging and PTT. The ability to utilize conventional ultrasound in combination with PA imaging enables the simultaneous acquisition of information about tumor morphology and micelle accumulation. PTT with CuNC(Octa)-loaded micelles can lead to the complete eradication of highly invasive tumors.

A commentary on 'Approved and investigational fluorescent optical imaging agents for disease detection in surgery'.

A commentary on 'Approved and investigational fluorescent optical imaging agents for disease detection in surgery'.

PET and Optical Imaging of Caveolin-1 in Gastric Tumors.

Previous studies have suggested tumoral caveolin-1 (CAV1) as a predictive biomarker for the response to anti-HER2 antibody drug therapies in gastric tumors. In this study, radiolabeled and fluorescently labeled anti-CAV1 antibodies were developed and tested as an immunoPET or optical imaging agent to detect CAV1 in HER2-positive/CAV1-high NCIN87 gastric tumors. The expression of CAV1 receptors in NCIN87 gastric tumors and nontumor murine organs was determined by Western blot. Binding assays were performed to validate the anti-CAV1 antibody specificity for CAV1-expressing NCIN87 cancer cells. Subcutaneous and orthotopic NCIN87 xenografts were used for PET imaging and ex vivo biodistribution of the radioimmunoconjugate. Additional HER2-PET and CAV1-optical imaging was also performed to determine CAV1 in the HER2-positive tumors. 89Zr-labeled anti-CAV1 antibody was able to bind to CAV1-expressing NCIN87 cells with a Bmax value of 2.7 × 103 CAV1 receptors/cell in vitro. ImmunoPET images demonstrated the localization of the antibody in subcutaneous NCIN87 xenografts. In the orthotopic model, CAV1 expression was also observed by optical imaging in the HER2-positive tumors previously imaged with HER2-PET. Ex vivo biodistribution analysis further confirmed these imaging results. The preclinical data from this study demonstrate the potential of using CAV1-PET and optical imaging for detecting gastric tumors.

Approved and investigational fluorescent optical imaging agents for disease detection in surgery.

Fluorescent optical imaging is becoming an increasingly attractive imaging tool that physicians can utilise as it can detect previously 'unseen' changes in tissue at a cellular level that are consistent with disease. This is possible using a range of fluorescently labelled imaging agents that, once excited by specific wavelengths of light, can illuminate damaged and diseased tissues. For surgeons, such agents can permit dynamic, intraoperative imaging providing a real-time guide as they resect diseased tissue.

Evaluation of nimotuzumab Fab2 as an optical imaging agent in EGFR positive cancers

Molecular-targeted imaging probes can be used with a variety of imaging modalities to detect diseased tissues and guide their removal. EGFR is a useful biomarker for a variety of cancers, because it is expressed at high levels relative to normal tissues. Previously, we showed the anti-EGFR antibody nimotuzumab can be used as a positron emission tomography and fluorescent imaging probe for EGFR positive cancers in mice. These imaging probes are currently in clinical trials for PET imaging and image-guided surgery, respectively. One issue with using antibody probes for imaging is their long circulation time and slow tissue penetration, which requires patients to wait a few days after injection before imaging or surgery, multiple visits and longer radiation exposure. Here, we generated a Fab2 fragment of nimotuzumab, by pepsin digestion and labeled it with IRDye800CW to evaluate its optical imaging properties. The Fab2 had faster tumor accumulation and clearance in mice relative to the nimotuzumab IgG. The fluorescent signal peaked at 2 h post injection and remained high until 6 h post injection. The properties of the Fab2 allow a higher signal to background to be obtained in a shorter time frame, reducing the wait time for imaging after probe infusion.

Optical Bioimaging and Therapy

Optical Bioimaging and Therapy

Multimodal Visualization of Bioorthogonal Systems by Off–On Luminescence and Enhanced Magnetic Resonance Imaging

AbstractBioorthogonal chemistry is a rising field investigating chemical reactions in physiological environments with high specificity. However, only very few examples concern the real‐time monitoring of bioorthogonal reactions by luminescence or magnetic relaxivity. To fill this gap, herein, the Eu(III)‐based complex is reported as a small‐molecule optical imaging agent which shows off–on luminescence and provides quantitative analysis for the progress of the bioorthogonal reaction. The characteristic signal is achieved through efficient energy harvesting and transferring to the Eu(III) from the expansion of the conjugated system of the antenna. Moreover, the gadolinium(III) counterpart significantly enhances relaxivity in magnetic resonance imaging (MRI) after the bioorthogonal reaction since the rotational correlation time is shortened with increased molecular sizes and weights.

Imaging Ligands Targeting Glypican-3 (GPC3) Receptor Expression in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and is increasing in incidence worldwide. Early detection of HCC is important since potentially curative therapies exist in the initial stages of HCC; no curative therapies exist for late-stage HCC. However, the initial detection of HCC remains challenging due to the lack of symptoms during the early stage of the disease. Other methods of screening and detecting HCC, including blood serum tests and conventional imaging methods, remain inadequate due to genetic differences between patients and the high background activity of liver tissues. Thus, there is a need for an accurate imaging agent for the diagnosis, staging, and prognosis of HCC. Glypican-3 (GPC3) is an oncofetal receptor responsible for regulating cell division, growth, and survival. GPC3 is a clinically relevant biomarker for imaging and therapeutics, as its expression is HCC tumor-specific and absent from normal and other pathological liver tissues. The development of novel GPC3-targeting imaging agents has encompassed three classes of biomolecules: peptides, antibodies, and aptamers. These biomolecules serve as constructs for diagnostic imaging (demonstrating potential as positron emission tomography [PET], single-photon emission tomography [SPECT], and optical imaging agents) and HCC treatment delivery. At least 20 unique ligands have been identified in the literature as showing specificity for the GPC3 receptor. Although several ligands are currently under clinical investigation as therapies for HCC, clinical translation of GPC3-targeting ligands as imaging agents is lacking. This review highlights the current landscape of ligands targeting GPC3 and describes their promising possibilities as imaging agents for HCC.

Polydimethylsiloxanes with Grafted Dibenzoylmethanatoboron Difluoride: Synthesis and Properties.

A method for the preparation of polydimethylsiloxanes with grafted methoxy-substituted dibenzoylmethanatoboron difluoride has been described. The structures of prepared polymers were confirmed using NMR, IR spectroscopy and gel permeation chromatography methods. Their thermal properties were investigated using thermal gravimetric analysis, differential scanning calorimetry and thermomechanical analysis. The prepared polymers had good thermal (Td5% up to 393 °C) and thermo-oxidative (Td5% = 413 °C) stability. The polymers started to transit in a viscous flow state at about 40 °C (for 3 a) and at about 20 °C (for 3 b). The viscoelastic characteristics of prepared polymers were determined in the sinusoidal oscillating vibrations mode. It was shown that the studied polymers at low frequencies at room temperature are viscoelastic fluids (G′ < G″). Increasing the frequency led to inversion (crossover) of dependences G′ and G″, which indicated the transition of polymers from viscous to elastomeric behavior characteristics, and the beginning of the formation of a physical network. Optical properties were studied using electron absorption, steady-state and time-resolved fluorescence spectroscopy. It was shown that intramolecular H-dimers exist in the ground state. The polymers studied had a bright fluorescence in the solution and in the solid state, consisting of bands of monomer and excimer emission. Thermally-activated delayed fluorescence was observed in the solution and the solid state. The prepared polymers possess intriguing properties that make them useful as optical materials, sensors or imaging agents.

Tuning the Solid- and Solution-State Fluorescence of the Iron-Chelator Deferasirox.

Deferasirox, an FDA-approved iron chelator, has gained increasing attention for use in anticancer and antimicrobial applications. Recent efforts by our group led to the identification of this core as an easy-to-visualize aggregation-induced emission platform, or AIEgen, that provides a therapeutic effect equivalent to deferasirox (J. Am. Chem. Soc. 2021, 143, 3, 1278-1283). However, the emission wavelength of the first-generation system overlapped with that of Syto9, a green emissive dye used to indicate live cells. Here, we report a library of deferasirox derivatives with various fluorescence emission profiles designed to overcome this limitation. We propose referring to systems that show promise as both therapeutic and optical imaging agents as "illuminoceuticals". The color differences between the derivatives were observable to the unaided eye (solid- and solution-state) and were in accord with the Commission Internationale de L'Eclairage (CIE) chromaticity diagram 1913. Each fluorescent derivative successfully imaged the respective spherical and rod shapes of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. They also displayed iron-dependent antibiotic activity. Three derivatives, ExNMe2 (3), ExTrisT (11), and ExDCM (13), display emission features that are sufficiently distinct so as to permit the multiplex (triplex) imaging of both MRSA and P. aeruginosa via stimulated emission depletion microscopy. The present deferasirox derivatives allowed for the construction of a multi-fluorophore sensor array. This array enabled the successful discrimination between Gram-positive/Gram-negative and drug-sensitive/drug-resistant bacteria. Antibiotic sensitivity and drug-resistant mutants from clinically isolated strains could also be identified and differentiated.

Optical Clearing Agents for Optical Imaging Through Cartilage Tympanoplasties: A Preclinical Feasibility Study.

Optical clearing agents (OCAs) can render cartilage tympanoplasty grafts sufficiently transparent to permit visualization of middle ear structures in an operated ear using optical coherence tomography (OCT) imaging. Pieces of human tragal cartilage were treated with glycerol, a commonly used OCA. A reference reflector was imaged with OCT through the tympanoplasty as it cleared and the optical attenuation of the graft was measured. The reversibility of clearing and the dimensional changes associated with glycerol absorption were also measured. In a separate experiment, a human cadaveric temporal bone was prepared to simulate an ossiculoplasty surgery with cartilage replacement of the tympanic membrane. A partial ossicular replacement prosthesis (PORP) inserted in the ear was imaged with OCT through a 0.4mm cartilage graft optically cleared with glycerol. The optical attenuation of 0.4mm cartilage grafts decreased at 2.3+/-1.1 dB/min following treatment with glycerol, reaching a total decrease in attenuation of 13.6+/-5.9 dB after 7 minutes. The optical and dimensional effects of glycerol absorption were reversable following saline washout. In the temporal bone preparation, treatment of a cartilage graft with glycerol resulted in a 13 dB increase in signal-to-noise ratio and a 13 dB increase in contrast for visualizing the PORP through the graft with OCT. Optical clearing agents offer a potential pathway towards optical coherence tomography imaging of the middle ear in post-surgical ears with cartilage grafts.

Real-Time Monitoring Renal Impairment Due to Drug-Induced AKI and Diabetes-Caused CKD Using an NAG-Activatable NIR-II Nanoprobe.

Real-time in vivo optical imaging of kidney function is important for the diagnosis of renal diseases, such as acute kidney injury (AKI) and chronic kidney disease (CKD), with high morbidity and mortality worldwide. However, the reported optical imaging agents still have limitations for identifying AKI or CKD in the early stage due to their low sensitivity, poor tissue penetration, and significant background interference. Herein, an N-acetyl-β-d-glucosaminidase (NAG)-activatable second near-infrared (NIR-II) fluorescent nanoprobe (BOD-II-NAG-NP) is developed for monitoring the progression of drug-induced AKI and in vivo imaging of diabetes-caused CKD. NAG, as a biomarker of renal diseases, is able to specifically activate BOD-II-NAG-NP to release NIR-II fluorescence signals, enabling in vivo imaging of kidney dysfunctions in living mice. Importantly, such an active imaging mechanism allows BOD-II-NAG-NP to noninvasively detect the onset of drug-induced AKI at least 32 h earlier than the most existing assays, which indicates that BOD-II-NAG-NP has the potential to be an optical imaging agent for the early diagnosis of AKI. Moreover, NIR-II fluorescence produced by BOD-II-NAG-NP could deeply penetrate into the relatively thick layers of fat in diabetic nephropathy mice and provide in vivo imaging with high resolution, indicating that BOD-II-NAG-NP has clinical potential for precision diagnosis of CKD.

Engineering of 177Lu-labeled gold encapsulated into dendrimeric nanomaterials for the treatment of lung cancer

As a novel type of theranostic radioactive agents, 177Lu-labeled nanomaterials conjugated to macromolecules have been described. The study aimed to fabricate PAMAM-G4–(177Lu–dendrimer)–bombesin–folate in the dendrimeric cavity, assess the radiopharmaceutical ability for specifically targeted radiotherapy and simultaneously detects gastrin-releasing peptide receptors (GRPR) and folate receptors (FRs) overexpressed in lung carcinoma cells, respectively. In an aqueous-basic media, p-SCN-benzyl-DOTA was conjugated to the dendrimer. This dendrimer was formed by activating the carboxylic acid groups of DOTA–folic acid and bombesin with HATU and conjugating them to develop the dendrimer. As part of this process, the conjugate was combined with 1% HAuCl4, added NaBH4 and filtered by ultrafiltration. Infrared, UV–Vis, TEM analysis, dynamic light scattering (DLS), and fluorescence spectroscopy were employed to observe the composition of the fabricated sample. Radio-labeled 177LuCl3 was used to label the conjugate, which was then evaluated using the radio-HPLC method. Findings demonstrated dendrimeric functionalization with remarkable radiochemical composition purity up to >96%. Because of fluorescence studies, it was determined that the occurrence of AuNMs in the dendrimeric cavities gives beneficial photo-physical characteristics to the radiopharmaceutical for bio-imaging. HEL-299 lung cancer cells exhibited a selective absorption of the drug (%). It might be helpful as nuclear and optical imaging agents for lung cancers that overexpress FRs and GRPR and as a specific target for radiation therapy if combined with folate–bombesin.

A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds

CD44 is emerging as an important receptor biomarker for various cancers. Amongst these is oral cancer, where surgical resection remains an essential mode of treatment. Unfortunately, surgery is frequently associated with permanent disfigurement, malnutrition, and functional comorbidities due to the difficultly of tumour removal. Optical imaging agents that can guide tumour tissue identification represent an attractive approach to minimising the impact of surgery. Here, we report the synthesis of a water-soluble fluorescent probe, namely HA-FA-HEG-OE (compound 1), that comprises components originating from natural sources: oleic acid, ferulic acid and hyaluronic acid. Compound 1 was found to be non-toxic, displayed aggregation induced emission and accumulated intracellularly in vesicles in SCC-9 oral squamous cells. The uptake of 1 was fully reversible over time. Internalization of compound 1 occurs through receptor mediated endocytosis; uniquely mediated through the CD44 receptor. Uptake is related to tumorigenic potential, with non-tumorigenic, dysplastic DOK cells and poorly tumorigenic MCF-7 cells showing only low intracellular levels and highlighting the critical role of endocytosis in cancer progression and metastasis. Together, the recognised importance of CD44 as a cancer stem cell marker in oral cancer, and the reversible, non-toxic nature of 1, makes it a promising agent for real time intraoperative imaging.

Near-Infrared Fluorescence In-Vivo Imaging of Non-Hodgkin Lymphoma Using Cy7-Bevacizumab

Introduction: Tumor angiogenesis is necessary for tumor growth and metastasis. Of the many known inducers that promote tumor angiogenesis, vascular endothelial growth factor (VEGF) plays the most important role. Increased VEGF expression has been found in many tumor types, including non-Hodgkin lymphoma (NHL). Bevacizumab is a humanized monoclonal antibody that recognizes all known isoforms of VEGF. This study aims to design a diagnostic molecular probe, Cy7-Bevacizumab, in which Cy7 is a near-infrared fluorescent dye designed to in-vivo non-invasively identify VEGF expression in NHL tumors.Methodology: VEGF expression was confirmed using flow cytometry on NHL cell line Toledo. Cy7-Bevacizumab was synthesized through nucleophilic substitution reaction between monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and Bevacizumab [1]. After purification, the conjugate was characterized by SDS-PAGE and spectrophotometry. For in-vivo imaging, Cy7-Bevacizumab (1 nmol) was injected intravenously in NHL tumor-bearing Balb/c nude mice (n=3) and was evaluated with near-infrared fluorescence (NIRF) at 2, 6, 24, 48, 72 and 96 h post-injection. After 96 h the lymph nodes accumulation was analyzed by ex-vivo NIRF images.Results: Flow cytometry showed that NHL Toledo cell line expresses high levels of VEGF. Bevacizumab was successfully labeled with Cy7, as shown by SDS-PAGE and spectrophotometry. Non-invasively NIRF in-vivo imaging of NHL tumor-bearing Balb/c nude mice allowed identification of tumors up to 96 h post-injection. Ex-vivo NIRF images confirmed tracer deposition at tumors.Conclusions: NHL Toledo cell line expresses VEGF. Cy7 labeled Bevacizumab has the potential to become an optical imaging agent for VEGF expressing tumors such as NHL, being possibly useful in guiding surgical excision of tumors and biopsies, which merits further evaluation.Acknowledgments: Agencia Nacional de Innovación e Investigación - Uruguay (ANII), Roche Laboratories, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay)) and Comisión Sectorial de Investigación Científica-Universidad de la República-Uruguay (CSIC, UdelaR) I+D Grupos Oncología Nuclear.Disclosures: No relevant conflicts of interest to declare.

A Standardized Framework for Fluorescence-Guided Margin Assessment for Head and Neck Cancer Using a Tumor Acidosis Sensitive Optical Imaging Agent

PurposeIntra-operative management of the surgical margin in patients diagnosed with head and neck squamous cell carcinoma (HNSCC) remains challenging as surgeons still have to rely on visual and tactile information. Fluorescence-guided surgery using tumor-specific imaging agents can assist in clinical decision-making. However, a standardized imaging methodology is lacking. In this study, we determined whether a standardized, specimen-driven, fluorescence imaging framework using ONM-100 could assist in clinical decision-making during surgery.ProceduresThirteen patients with histologically proven HNSCC were included in this clinical study and received ONM-100 24 ± 8 h before surgery. Fluorescence images of the excised surgical specimen and of the surgical cavity were analyzed. A fluorescent lesion with a tumor-to-background ratio (TBR) > 1.5 was considered fluorescence-positive and correlated to standard of care (SOC) histopathology.ResultsAll six tumor-positive surgical margins were detected immediately after excision using fluorescence-guided intra-operative imaging. Postoperative analysis showed a median TBR (±IQR) of the fluorescent lesions on the resection margin of 3.36 ± 1.62. Three fluorescence-positive lesions in the surgical cavity were biopsied and showed occult carcinoma and severe dysplasia, and a false-positive fluorescence lesion.ConclusionOur specimen-driven fluorescence framework using a novel, pH-activatable, fluorescent imaging agent could assist in reliable and real-time adequate clinical decision-making showing that a fluorescent lesion on the surgical specimen with a TBR of 1.5 is correlated to a tumor-positive resection margin. The binary mechanism of ONM-100 allows for a sharp tumor delineation in all patients, giving the surgeon a clinical tool for real-time margin assessment, with a high sensitivity.

5-Aminolevulinic Acid as a Novel Therapeutic for Inflammatory Bowel Disease.

5-Aminolevulinic acid (5-ALA) is a naturally occurring nonprotein amino acid licensed as an optical imaging agent for the treatment of gliomas. In recent years, 5-ALA has been shown to possess anti-inflammatory and immunoregulatory properties through upregulation of heme oxygenase-1 via enhancement of porphyrin, indicating that it may be beneficial for the treatment of inflammatory conditions. This study systematically examines 5-ALA for use in inflammatory bowel disease (IBD). Firstly, the ex vivo colonic stability and permeability of 5-ALA was assessed using human and mouse fluid and tissue. Secondly, the in vivo efficacy of 5-ALA, in the presence of sodium ferrous citrate, was investigated via the oral and intracolonic route in an acute DSS colitis mouse model of IBD. Results showed that 5-ALA was stable in mouse and human colon fluid, as well as in colon tissue. 5-ALA showed more tissue restricted pharmacokinetics when exposed to human colonic tissue. In vivo dosing demonstrated significantly improved colonic inflammation, increased local heme oxygenase-1 levels, and decreased concentrations of proinflammatory cytokines TNF-α, IL-6, and IL-1β in both plasma and colonic tissue. These effects were superior to that measured concurrently with established anti-inflammatory treatments, ciclosporin and 5-aminosalicylic acid (mesalazine). As such, 5-ALA represents a promising addition to the IBD armamentarium, with potential for targeted colonic delivery.