Albumin-seeking dyes with adjustable assemblies in situ enable programmable imaging windows and targeting tumor imaging.

Cyanine dyes are widely used organic probes for in vivo imaging due to their tunable fluorescence. They can form complexes with endogenous albumin, resulting in enhanced brightness and photostability. However, this binding is uncontrollable and irreversible, leading to considerable nonspecific background signals and unregulated circulation time. Methods: Here, we connect varying numbers of 4-(4-iodophenyl) butanoic acid (IP) as albumin-binding moieties (ABM) to the cyanine dye, enabling dynamic and controllable binding with albumin. Meanwhile, we provide a blocking method to completely release the dye from covalent capture with albumin, resulting in specific targeting fluorescence. Furthermore, we evaluate the pharmacokinetics and tumor targeting of the developed dyes. Results: The engineered dyes can dynamically and selectively bind with multiple albumins to change the in situ size of assemblies and circulation time, providing programmable regulation over the imaging time window. The nucleophilic substitution of meso-Cl with water-soluble amino acids or targeting peptides for IP-engineered dye further addresses the nonspecific signals caused by albumin, allowing for adjustable angiography time and efficient tumor targeting. Conclusion: This study rationalizes the binding modes of dyes and proteins, applicable to a wide range of near-infrared (NIR) dyes for improving their in vivo molecular imaging.

Improved pharmacokinetic and lymphatic uptake of Rose Bengal after transfersome intradermal deposition using hollow microneedles.

The lymphatic system is active in several processes that regulate human diseases, among which cancer progression stands out. Thus, various drug delivery systems have been investigated to promote lymphatic drug targeting for cancer therapy; mainly, nanosized particles in the 10-150 nm range quickly achieve lymphatic vessels after an interstitial administration. Herein, a strategy to boost the lymphotropic delivery of Rose Bengal (RB), a hydrosoluble chemotherapeutic, is proposed, and it is based on the loading into Transfersomes (RBTF) and their intradermal deposition in vivo by microneedles. RBTF of 96.27 ± 13.96 nm (PDI = 0.29 ± 0.02) were prepared by a green reverse-phase evaporation technique, and they showed an RB encapsulation efficiency of 98.54 ± 0.09%. In vitro, RBTF remained physically stable under physiological conditions and avoided the release of RB. In vivo, intravenous injection of RBTF prolonged RB half-life of 50 min in healthy rats compared to RB intravenous injection; the RB half-life in rat body was further increased after intradermal injection reaching 24 h, regardless of the formulation used. Regarding lymphatic targeting, RBTF administered intravenously provided an RB accumulation in the lymph nodes of 12.3 ± 0.14 ng/mL after 2 h, whereas no RB accumulation was observed after RB intravenous injection. Intradermally administered RBTF resulted in the highest RB amount detected in lymph nodes after 2 h from the injection (84.2 ± 25.10 ng/mL), which was even visible to the naked eye based on the pink colouration of the drug. In the case of intradermally administered RB, RB in lymph node was detected only at 24 h (13.3 ± 1.41 ng/mL). In conclusion, RBTF proved an efficient carrier for RB delivery, enhancing its pharmacokinetics and promoting lymph-targeted delivery. Thus, RBTF represents a promising nanomedicine product for potentially facing the medical need for novel strategies for cancer therapy.

Ultrabright Renal-Clearable Cyanine-Protein Nanoprobes for High-Quality NIR-II Angiography and Lymphography.

The renal-clearable aspect of imaging agent with minimum toxicity issues and side effects is essential for clinical translation, yet clinical near-infrared-I/II (NIR-I/II) fluorophores with timely renal-clearance pathways are very limited. Herein, we rationally develop the cyanine-protein composite strategy through covalent bonding of ß-lactoglobulin (ß-LG) and chloride-cyanine dye to produce a brilliant and stable NIR-I/II fluorophore (e.g., ß-LG@IR-780). The ß-LG acts as a protecting shell with small molecular weight (18.4 kDa) and ultrasmall size (<5 nm), thus endowing the ß-LG@IR-780 with excellent biocompatibility and renal excretion. Our ß-LG@IR-780 probe enables noninvasive and precise NIR-II visualization of the physiological and pathological conditions of the vascular and lymphatic drainage system, facilitating intraoperative imaging-guided surgery and postoperative noninvasive monitoring. The minimum accumulation of our probes in the main organs improves the overall biosafety. This study provides a facile methodology for new-generation NIR-II fluorophores and largely improves the brightness and pharmacokinetics of small molecular dyes.

In vivo accurate detection of the liver tumor with pharmacokinetic parametric images from dynamic fluorescence molecular tomography.

SIGNIFICANCE: Pharmacokinetic parametric images in dynamic fluorescence molecular tomography (FMT) can describe three-dimensional (3D) physiological and pathological information inside biological tissues, potentially providing quantitative assessment tools for biological research and drug development. AIM: In vivo imaging of the liver tumor with pharmacokinetic parametric images from dynamic FMT based on the differences in metabolic properties of indocyanine green (ICG) between normal liver cells and tumor liver cells inside biological tissues. APPROACH: First, an orthotopic liver tumor mouse model was constructed. Then, with the help of the FMT/computer tomography (CT) dual-modality imaging system and the direct reconstruction algorithm, 3D imaging of liver metabolic parameters in nude mice was achieved to distinguish liver tumors from normal tissues. Finally, pharmacokinetic parametric imaging results were validated against in vitro anatomical results. RESULTS: This letter demonstrates the ability of dynamic FMT to monitor the pharmacokinetic delivery of the fluorescent dye ICG in vivo, thus, enabling the distinction between normal and tumor tissues based on the pharmacokinetic parametric images derived from dynamic FMT. CONCLUSIONS: Compared with CT structural imaging technology, dynamic FMT combined with compartmental modeling as an analytical method can obtain quantitative images of pharmacokinetic parameters, thus providing a more powerful research tool for organ function assessment, disease diagnosis and new drug development.

Guest-host doped strategy for constructing ultralong-lifetime near-infrared organic phosphorescence materials for bioimaging.

Organic near-infrared room temperature phosphorescence materials have unparalleled advantages in bioimaging due to their excellent penetrability. However, limited by the energy gap law, the near-infrared phosphorescence materials (>650 nm) are very rare, moreover, the phosphorescence lifetimes of these materials are very short. In this work, we have obtained organic room temperature phosphorescence materials with long wavelengths (600/657-681/732 nm) and long lifetimes (102-324 ms) for the first time through the guest-host doped strategy. The guest molecule has sufficient conjugation to reduce the lowest triplet energy level and the host assists the guest in exciton transfer and inhibits the non-radiative transition of guest excitons. These materials exhibit good tissue penetration in bioimaging. Thanks to the characteristic of long lifetime and long wavelength emissive phosphorescence materials, the tumor imaging in living mice with a signal to background ratio value as high as 43 is successfully realized. This work provides a practical solution for the construction of organic phosphorescence materials with both long wavelengths and long lifetimes.

Photoactivatable Fluorescent Tags for Dual-Modality Positron Emission Tomography Optical Imaging.

Fluorescent protein conjugates are vital tools in a wide range of scientific disciplines from basic biochemical research to applications in clinical pathology and intraoperative surgery. We report the synthesis and characterization of photoactivatable fluorophores (PhotoTags) based on the functionalization of coumarin, fluorescein, BODIPY, rhodamine B, and cyanine dyes with a photochemically active aryl azide group. Photochemical labeling experiments using human serum albumin produced fluorescent proteins in high yields under irradiation with ultraviolet light for <15 min. We also synthesized DFO-RhodB-PEG3-ArN3─a photoactivatable compound that can be radiolabeled with 89Zr for applications in optical imaging and positron emission tomography. One-pot 89Zr-radiolabeling and light-induced protein conjugation produced [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab. Proof-of-concept studies in vitro and in vivo confirmed that [89Zr]ZrDFO-RhodB-PEG3-azepin-trastuzumab is a potential dual-modality agent for detecting human epidermal growth factor receptor 2 (HER2/neu) expression. Overall, the PhotoTag technology represents a rapid, synthetically versatile, and user-friendly approach for generating novel protein conjugates.

Self-assembled NIR-II Fluorophores with Ultralong Blood Circulation for Cancer Imaging and Image-guided Surgery.

Complete excision of the last remaining 1-2% of tumor tissue without collateral damage remains particularly challenging. Herein, we report thiophenthiadiazole (TTD)-derived fluorophores L6-PEGnk (n = 1, 2, 5) as new-generation NIR-II (1000-1700 nm) probes with exceptional nonfouling performance and significantly high fluorescence quantum yields in water. L6-PEG2k can self-assemble into vesicular micelles and exhibited minimal immunogenicity, low binding affinities, ultralong blood circulation (t1/2 = 59.5 h), and a supercontrast ratio in vivo. Most importantly, L6-PEG2k achieved excellent in vivo CT-26 and U87MG tumor targeting and accumulation (>20 d) through intraperitoneal or intravenous injection. A subcutaneous U87MG tumor and orthotopic brain glioma were successfully resected under NIR-II FIGS in our animal model via intraperitoneal injection in an extended time window (48-144 h). This study highlights the potential of using L6-PEG2K as self-assembling molecular probes with long-circulation persistence for routine preoperative tumor assessment and precise intraoperative image-guided resection.

4-Azidocinnoline-Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe.

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide-amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.

Rational Design of Surface-State Controlled Multicolor Cross-Linked Carbon Dots with Distinct Photoluminescence and Cellular Uptake Properties.

We disclose for the first time a facile synthetic methodology for the preparation of multicolor carbon dots (CDs) from a single source barring any chromatographic separations. This was achieved via sequential intraparticle cross-linking of surface abundant carboxylic acid groups on the CDs synthesized from a precursor to control their photoluminescence (PL) spectra as well as affect their degree of cellular internalization in cancer cells. The change in PL spectra with sequential cross-linking was projected by theoretical density functional theory (DFT) studies and validated by multiple characterization tools such as X-ray photoelectron spectroscopy (XPS), PL spectroscopy, ninhydrin assay, etc. The variation in cellular internalization of these cross-linked CDs was demonstrated using inhibitor assays, confocal microscopy, and flow cytometry. We supplemented our findings with high-resolution dark-field imaging to visualize and confirm the colocalization of these CDs into distinct intracellular compartments. Finally, to prove the surface-state controlled PL mechanisms of these cross-linked CDs, we fabricated a triple-channel sensor array for the identification of different analytes including metal ions and biologically relevant proteins.

Dye-loaded mesoporous polydopamine nanoparticles for multimodal tumor theranostics with enhanced immunogenic cell death.

BACKGROUND: Tumor phototherapy especially photodynamic therapy (PDT) or photothermal therapy (PTT), has been considered as an attractive strategy to elicit significant immunogenic cell death (ICD) at an optimal tumor retention of PDT/PTT agents. Heptamethine cyanine dye (IR-780), a promising PDT/PTT agent, which can be used for near-infrared (NIR) fluorescence/photoacoustic (PA) imaging guided tumor phototherapy, however, the strong hydrophobicity, short circulation time, and potential toxicity in vivo hinder its biomedical applications. To address this challenge, we developed mesoporous polydopamine nanoparticles (MPDA) with excellent biocompatibility, PTT efficacy, and PA imaging ability, facilitating an efficient loading and protection of hydrophobic IR-780. RESULTS: The IR-780 loaded MPDA (IR-780@MPDA) exhibited high loading capacity of IR-780 (49.7 wt%), good physiological solubility and stability, and reduced toxicity. In vivo NIR fluorescence and PA imaging revealed high tumor accumulation of IR-780@MPDA. Furthermore, the combined PDT/PTT of IR-780@MPDA could induce ICD, triggered immunotherapeutic response to breast tumor by the activation of cytotoxic T cells, resulting in significant suppression of tumor growth in vivo. CONCLUSION: This study demonstrated that the as-developed compact and biocompatible platform could induce combined PDT/PTT and accelerate immune activation via excellent tumor accumulation ability, offering multimodal tumor theranostics with negligible systemic toxicity.

Rapidly liver-clearable rare-earth core-shell nanoprobe for dual-modal breast cancer imaging in the second near-infrared window.

BACKGROUND: Fluorescence imaging as the beacon for optical navigation has wildly developed in preclinical studies due to its prominent advantages, including noninvasiveness and superior temporal resolution. However, the traditional optical methods based on ultraviolet (UV, 200-400 nm) and visible light (Vis, 400-650 nm) limited by their low penetration, signal-to-noise ratio, and high background auto-fluorescence interference. Therefore, the development of near-infrared-II (NIR-II 1000-1700 nm) nanoprobe attracted significant attentions toward in vivo imaging. Regrettably, most of the NIR-II fluorescence probes, especially for inorganic NPs, were hardly excreted from the reticuloendothelial system (RES), yielding the anonymous long-term circulatory safety issue. RESULTS: Here, we develop a facile strategy for the fabrication of Nd3+-doped rare-earth core-shell nanoparticles (Nd-RENPs), NaGdF4:5%Nd@NaLuF4, with strong emission in the NIR-II window. What's more, the Nd-RENPs could be quickly eliminated from the hepatobiliary pathway, reducing the potential risk with the long-term retention in the RES. Further, the Nd-RENPs are successfully utilized for NIR-II in vivo imaging and magnetic resonance imaging (MRI) contrast agents, enabling the precise detection of breast cancer. CONCLUSIONS: The rationally designed Nd-RENPs nanoprobes manifest rapid-clearance property revealing the potential application toward the noninvasive preoperative imaging of tumor lesions and real-time intra-operative supervision.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

A Novel NIR Fluorescent Probe for Highly Selective Detection of Nitroreductase and Hypoxic-Tumor-Cell Imaging.

Nitroreductase as a potential biomarker for aggressive tumors has received extensive attention. In this work, a novel NIR fluorescent probe for nitroreductase detection was synthesized. The probe Py-SiRh-NTR displayed excellent sensitivity and selectivity. Most importantly, the confocal fluorescence imaging demonstrated that HepG-2 cells treated with Py-SiRh-NTR under hypoxic conditions showed obvious enhanced fluorescence, which means that the NTR was overexpressed under hypoxic conditions. Moreover, the probe showed great promise that could help us to study related anticancer mechanisms research.

Tetrahydrocannabinol and Its Major Metabolites Are Not (or Are Poor) Substrates or Inhibitors of Human P-Glycoprotein [ATP-Binding Cassette (ABC) B1] and Breast Cancer Resistance Protein (ABCG2).

(-)-Δ9-Tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis. In humans, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH) are psychoactive and nonpsychoactive circulating metabolites of THC, respectively. Whether these cannabinoids are substrates or inhibitors of human P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) is unknown. Previous animal studies suggest that THC and its metabolites could be substrates of these transporters. Therefore, we performed Transwell, cellular accumulation, and vesicular transport assays, at pharmacologically relevant concentrations of these cannabinoids, using Madin-Darby canine kidney (MDCK) II cells or plasma membrane vesicles overexpressing human P-gp or BCRP. Neither THC nor 11-OH-THC was found to be a substrate or inhibitor of P-gp or BCRP. The efflux ratio of THC-COOH in MDCKII-BCRP cells was 1.6, which was significantly decreased to 1.0 by the BCRP inhibitor Ko143. Likewise, cellular accumulation of THC-COOH was significantly increased 1.6-fold in the presence versus absence of Ko143. THC-COOH also significantly inhibited BCRP-mediated transport of Lucifer yellow, a BCRP substrate; however, THC-COOH was neither a substrate nor an inhibitor of P-gp. Collectively, these results indicate that THC and 11-OH-THC are not substrates or inhibitors (at pharmacologically relevant concentrations) of either P-gp or BCRP. THC-COOH is a weak substrate and inhibitor of BCRP, but not of P-gp. Accordingly, we predict that P-gp/BCRP will not modulate the disposition of these cannabinoids in humans. In addition, use of these cannabinoids will not result in P-gp- or BCRP-based drug interactions. SIGNIFICANCE STATEMENT: This study systematically investigated whether Δ9-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-THC and 11-nor-9-carboxy-THC, are substrates and/or inhibitors of human P-gp and BCRP at pharmacologically relevant concentrations. The results obtained are highly valuable for mechanistic understanding and prediction of the roles of P-gp and BCRP in determining the human pharmacokinetics, tissue distribution, and drug interactions of cannabinoids.

Imaging-Guided Chemo-Photothermal Polydopamine Carbon Dots for EpCAM-Targeted Delivery toward Liver Tumor.

We demonstrate a versatile nanoparticle with imaging-guided chemo-photothermal synergistic therapy and EpCAM-targeted delivery of liver tumor cells. EpCAM antibody (anti-EpCAM) and Pt(IV) were grafted onto the polydopamine carbon dots (PDA-CDs) by the amidation reaction. The EpCAM antibody of particles enables the targeted interaction with liver progenitor cells due to their overexpressed EpCAM protein. The tetravalent platinum prodrug [Pt(IV)] induces apoptosis with minimum toxic side effects through the interaction between cisplatin and tumor cell DNA. The nanoparticles displayed stable photothermal property and considerable anti-tumor therapeutic effect in vivo. Coupling with cellular imaging due to their fluorescence property, anti-EpCAM@PDA-CDs@Pt(IV) offers a convenient and effective platform for imaging-guided chemo-photothermal synergistic therapy toward liver cancers in the near future.

A mitochondria-targeted near-infrared fluorescent probe for imaging viscosity in living cells and a diabetic mice model.

A mitochondria-targeted near-infrared fluorescent probe NIR-V with 700 nm emission was designed to monitor cell viscosity changes with high selectivity and sensitivity, which was applied to detect the intracellular viscosity and image pancreatic tissue in a diabetic mouse model. Probe NIR-V provides an effective way to diagnose viscosity related diseases.

Carbohydrate based biomarkers enable hybrid near infrared fluorescence and 64Cu based radio-guidance for improved surgical precision.

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64Cu for 48 hours. The retention and biodistribution of 64Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions.

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response.

PURPOSE: Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking. PROCEDURE: We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples. RESULTS: We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression. CONCLUSION: Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer.

Indocyanine green (ICG) is a near infrared fluorescent tracer used in image-guided surgery to assist surgeons during resection. Despite appearing as a very promising tool for surgical oncology, its employment in this area is limited to lymph node mapping or to laparoscopic surgery, as it lacks tumor targeting specificity. Recently, a nanoformulation of this dye has been proposed with the aim toward tumor targeting specificity in order to expand its employment in surgical oncology. This nanosystem is constituted by 24 monomers of H-Ferritin (HFn), which self-assemble into a spherical cage structure enclosing the indocyanine green fluorescent tracer. These HFn nanocages were demonstrated to display tumor homing due to the specific interaction between the HFn nanocage and transferrin receptor 1, which is overexpressed in most tumor tissues. Here, we provide an ex vivo detailed comparison between the biodistribution of this nanotracer and free ICG, combining the results obtained with the Karl Storz endoscope that is currently used in clinical practice and the quantification of the ICG signal derived from the fluorescence imaging system IVIS Lumina II. These insights demonstrate the suitability of this novel HFn-based nanosystem in fluorescence-guided oncological surgery.

Transfer of rhodamine-123 into the brain and cerebrospinal fluid of fetal, neonatal and adult rats.

BACKGROUND: Adenosine triphosphate binding cassette transporters such as P-glycoprotein (PGP) play an important role in drug pharmacokinetics by actively effluxing their substrates at barrier interfaces, including the blood-brain, blood-cerebrospinal fluid (CSF) and placental barriers. For a molecule to access the brain during fetal stages it must bypass efflux transporters at both the placental barrier and brain barriers themselves. Following birth, placental protection is no longer present and brain barriers remain the major line of defense. Understanding developmental differences that exist in the transfer of PGP substrates into the brain is important for ensuring that medication regimes are safe and appropriate for all patients. METHODS: In the present study PGP substrate rhodamine-123 (R123) was injected intraperitoneally into E19 dams, postnatal (P4, P14) and adult rats. Naturally fluorescent properties of R123 were utilized to measure its concentration in blood-plasma, CSF and brain by spectrofluorimetry (Clariostar). Statistical differences in R123 transfer (concentration ratios between tissue and plasma ratios) were determined using Kruskal-Wallis tests with Dunn's corrections. RESULTS: Following maternal injection the transfer of R123 across the E19 placenta from maternal blood to fetal blood was around 20 %. Of the R123 that reached fetal circulation 43 % transferred into brain and 38 % into CSF. The transfer of R123 from blood to brain and CSF was lower in postnatal pups and decreased with age (brain: 43 % at P4, 22 % at P14 and 9 % in adults; CSF: 8 % at P4, 8 % at P14 and 1 % in adults). Transfer from maternal blood across placental and brain barriers into fetal brain was approximately 9 %, similar to the transfer across adult blood-brain barriers (also 9 %). Following birth when placental protection was no longer present, transfer of R123 from blood into the newborn brain was significantly higher than into adult brain (3 fold, p < 0.05). CONCLUSIONS: Administration of a PGP substrate to infant rats resulted in a higher transfer into the brain than equivalent doses at later stages of life or equivalent maternal doses during gestation. Toxicological testing of PGP substrate drugs should consider the possibility of these patient specific differences in safety analysis.