Nanotherapeutic kidney cell-specific targeting to ameliorate acute kidney injury

Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney. Acute kidney injury (AKI) increases the risk of in-hospital death, adds to expense of care, and risk of early chronic kidney disease. AKI often follows an acute event such that timely treatment could ameliorate AKI and potentially reduce the risk of additional disease. Despite therapeutic success of dexamethasone in animal models, clinical trials have not demonstrated broad success. To improve the safety and efficacy of dexamethasone for AKI, we developed and characterized a novel, kidney-specific nanoparticle enabling specific within-kidney targeting to proximal tubular epithelial cells provided by the megalin ligand cilastatin. Cilastatin and dexamethasone were complexed to H-Dot nanoparticles, which were constructed from generally recognized as safe components. Cilastatin/Dexamethasone/H-Dot nanotherapeutics were found to be stable at plasma pH and demonstrated salutary release kinetics at urine pH. In vivo, they were specifically biodistributed to the kidney and bladder, with 75% recovery in the urine and with reduced systemic toxicity compared to native dexamethasone. Cilastatin complexation conferred proximal tubular epithelial cell specificity within the kidney in vivo and enabled dexamethasone delivery to the proximal tubular epithelial cell nucleus in vitro. The Cilastatin/Dexamethasone/H-Dot nanotherapeutic improved kidney function and reduced kidney cellular injury when administered to male C57BL/6 mice in two translational models of AKI (rhabdomyolysis and bilateral ischemia reperfusion). Thus, our design-based targeting and therapeutic loading of a kidney-specific nanoparticle resulted in preservation of the efficacy of dexamethasone, combined with reduced off-target disposition and toxic effects. Hence, our study illustrates a potential strategy to target AKI and other diseases of the kidney. Translational StatementAcute kidney injury complicates severe illness and can lead to death or chronic kidney disease. One challenge for treatment is targeting the kidney. Here we specifically target the injured cell population with a kidney-specific nanoparticle that contains a targeting moiety and dexamethasone as a treatment moiety. The nanoparticle has a generally recognized as safe backbone and is complexed to US Food and Drug Administration–approved targeting and therapeutic moieties, reducing barriers to translation. Kidney-specific delivery and reduction of toxicity increase utility in the complex environment of care around acute kidney injury. These data support the rodent and large animal translational study and scale-up pharmaceutical-grade production to support phase I trials. Acute kidney injury complicates severe illness and can lead to death or chronic kidney disease. One challenge for treatment is targeting the kidney. Here we specifically target the injured cell population with a kidney-specific nanoparticle that contains a targeting moiety and dexamethasone as a treatment moiety. The nanoparticle has a generally recognized as safe backbone and is complexed to US Food and Drug Administration–approved targeting and therapeutic moieties, reducing barriers to translation. Kidney-specific delivery and reduction of toxicity increase utility in the complex environment of care around acute kidney injury. These data support the rodent and large animal translational study and scale-up pharmaceutical-grade production to support phase I trials. Acute kidney injury (AKI) is a lethal syndrome caused by traumatic injury, surgery, coronavirus disease 2019, critical illness, and therapeutic drugs. In these settings, the time of kidney injury is often known, and timely treatment could ameliorate AKI and prevent additional disease. AKI increases hospital length of stay, risk of in-hospital death, and expense of care.1Dasta J.F. Kane-Gill S. Review of the literature on the costs associated with acute kidney injury.J Pharm Pract. 2019; 32: 292-302Crossref PubMed Scopus (35) Google Scholar,2Silver S.A. Long J. Zheng Y. et al.Cost of acute kidney injury in hospitalized patients.J Hosp Med. 2017; 12: 70-76Crossref PubMed Scopus (154) Google Scholar The risk of transition to chronic kidney disease in patients with AKI older than 66 years is 50%,3United States Renal Data System 2020 USRDS Annual Data Report: epidemiology of kidney disease in the United States.National Institute of Diabetes and Digestive and Kidney Diseases. National Institutes of Health, 2000Google Scholar and there is a considerable increase in the risk of distant organ disease, as well.4Hebert J.F. Funahashi Y. Hutchens M.P. Harm! foul! How acute kidney injury SHReDDs patient futures.Curr Opin Nephrol Hypertens. 2023; 32: 165-171Crossref PubMed Scopus (1) Google Scholar Despite success in animal models, clinical trials have failed to demonstrate a safe therapeutic agent. A major reason for such failures is pleiotropic extrarenal effects of systemically delivered agents. For example, the anti-inflammatory glucocorticoid dexamethasone (Dex) is protective in mice subjected to renal ischemia-reperfusion (a model of early graft dysfunction after renal transplantation) and experimental rhabdomyolysis (a model of traumatic crush syndrome).5Kumar S. Allen D.A. Kieswich J.E. et al.Dexamethasone ameliorates renal ischemia-reperfusion injury.J Am Soc Nephrol. 2009; 20: 2412-2425Crossref PubMed Scopus (101) Google Scholar, 6Zager R.A. Johnson A.C.M. Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.Am J Physiol Renal Physiol. 2019; 316: F674-F681Crossref PubMed Scopus (14) Google Scholar, 7Moonen L. Geryl H. D'Haese P.C. et al.Short-term dexamethasone treatment transiently, but not permanently, attenuates fibrosis after acute-to-chronic kidney injury.BMC Nephrol. 2018; 19: 343Crossref PubMed Scopus (11) Google Scholar Dex has been administered to patients undergoing cardiac surgery, a setting with a high risk of AKI and in which severe side effects of Dex (immunosuppression, hyperglycemia, and delirium) can be managed in the intensive care unit. In the largest and best-conducted clinical study, Dex reduced the risk of severe AKI, especially in high-risk patients.8Jacob K.A. Leaf D.E. Dieleman J.M. et al.Intraoperative high-dose dexamethasone and severe AKI after cardiac surgery.J Am Soc Nephrol. 2015; 26: 2947-2951Crossref PubMed Scopus (74) Google Scholar However, severe side effects of Dex, including delirium, hyperglycemia, hypertension, muscle catabolism, and immunosuppression,9Dexamethasone. Package insert. Acrotech Biopharma, 2019Google Scholar prevent its wide use for AKI. Although Dex has pleiotropic anti-inflammatory and immune suppressant properties, Dex protects cultured proximal tubular epithelial cell (PTEC) survival, morphology, and mitochondrial function in injury models, suggesting a specific effect on PTEC.6Zager R.A. Johnson A.C.M. Acute kidney injury induces dramatic p21 upregulation via a novel, glucocorticoid-activated, pathway.Am J Physiol Renal Physiol. 2019; 316: F674-F681Crossref PubMed Scopus (14) Google Scholar,10Schirris T.J.J. Jansen J. Mihajlovic M. et al.Mild intracellular acidification by dexamethasone attenuates mitochondrial dysfunction in a human inflammatory proximal tubule epithelial cell model.Sci Rep. 2017; 7: 10623Crossref PubMed Scopus (4) Google Scholar,11Zager R.A. Johnson A.C. Becker K. Acute unilateral ischemic renal injury induces progressive renal inflammation, lipid accumulation, histone modification, and "end-stage" kidney disease.Am J Physiol Renal Physiol. 2011; 301: F1334-F1345Crossref PubMed Scopus (134) Google Scholar AKI-related tubular cell cycle arrest mediates recovery, repair, and development of AKI–chronic kidney disease transition.12Lin X. Jin H. Chai Y. et al.Cellular senescence and acute kidney injury.Pediatr Nephrol. 2022; 37: 3009-3018Crossref Scopus (19) Google Scholar,13De Chiara L. Conte C. Antonelli G. et al.Tubular cell cycle response upon AKI: revising old and new paradigms to identify novel targets for CKD prevention.Int J Mol Sci. 2021; 22: 11093Crossref Scopus (18) Google Scholar Because it is proven in humans, control of the pharmacokinetics and pharmacodynamics of Dex to prevent toxicity would enable its use as a treatment. We previously reported development of multifunctional nanocarriers, on the basis of a generally regarded as safe backbone, with controllable substitution sites, which enable targeting, imaging, and image-guided interventions.14Kang H. Gravier J. Bao K. et al.Renal clearable organic nanocarriers for bioimaging and drug delivery.Adv Mater. 2016; 28: 8162-8168Crossref PubMed Scopus (113) Google Scholar Because of size and charge characteristics, these zwitterionic nanocarriers, termed "H-Dots," have profound specificity for the renal filtrate in addition to salutary near-infrared (NIR) fluorescence (for targeting studies) and lack of association with serum proteins, liver, brain, or other off-target tissues.14Kang H. Gravier J. Bao K. et al.Renal clearable organic nanocarriers for bioimaging and drug delivery.Adv Mater. 2016; 28: 8162-8168Crossref PubMed Scopus (113) Google Scholar,15Kang H. Han M. Xue J. et al.Renal clearable nanochelators for iron overload therapy.Nat Commun. 2019; 10: 5134Crossref PubMed Scopus (98) Google Scholar Therefore, we hypothesized that H-Dot nanocarriers, already kidney specific, could be customized to superspecifically target them within the kidney to tubular epithelial cells (PTECs) and deliver Dex only to these cells, ameliorating AKI and preventing Dex-related toxicities. To target PTEC, we used a high-affinity megalin ligand, cilastatin (Cil) sodium.16Hori Y. Aoki N. Kuwahara S. et al.Megalin blockade with cilastatin suppresses drug-induced nephrotoxicity.J Am Soc Nephrol. 2017; 28: 1783-1791Crossref PubMed Scopus (99) Google Scholar Cil has a low toxicity and is approved by the US Food and Drug Administration in combination with the antibiotic imipenem.17U.S. Food and Drug Administration. Primaxin (imipenem/cilastatin sodium). FDA briefing document.https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/050587s074lbl.pdfGoogle Scholar In addition, studies suggest that at high concentration Cil may have renoprotective properties in injury models.18Matsushita K. Mori K. Saritas T. et al.Cilastatin ameliorates rhabdomyolysis-induced AKI in mice.J Am Soc Nephrol. 2021; 32: 2579-2594Crossref PubMed Scopus (25) Google Scholar, 19Gonzalez-Fernandez R. Gonzalez-Nicolas M.A. Morales M. et al.FKBP51, AmotL2 and IQGAP1 involvement in cilastatin prevention of cisplatin-induced tubular nephrotoxicity in rats.Cells. 2022; 11: 1585Crossref Scopus (2) Google Scholar, 20Humanes B. Jado J.C. Camano S. et al.Protective effects of cilastatin against vancomycin-induced nephrotoxicity.Biomed Res Int. 2015; 2015: 704382Crossref PubMed Scopus (44) Google Scholar, 21Jado J.C. Humanes B. Gonzalez-Nicolas M.A. et al.Nephroprotective effect of cilastatin against gentamicin-induced renal injury in vitro and in vivo without altering its bactericidal efficiency.Antioxidants (Basel). 2020; 9: 821Crossref Scopus (23) Google Scholar, 22Zaballos M. Power M. Canal-Alonso M.I. et al.Effect of cilastatin on cisplatin-induced nephrotoxicity in patients undergoing hyperthermic intraperitoneal chemotherapy.Int J Mol Sci. 2021; 22: 1239Crossref Scopus (11) Google Scholar To test our hypothesis, we developed Cil/Dex/H-Dot and characterized its pharmacokinetics and dynamics. Primary culture of human tubular epithelial cells was used to test the mechanism of targeting, payload delivery, and drug action. Finally, efficacy was tested in vivo in translational AKI models. Detailed methods are provided in Supplementary Methods. Briefly, monoaldehyde β-cyclodextrin is prepared and conjugated onto the ε-polylysine (EPL) backbone to construct cyclodextrin polylysine. Next, the fluorophore ZW800-1 is conjugated to CDPL. The resulting ZW800-CDPL is succinylated and dialyzed, producing a complexable H-Dot nanoparticle. Lastly, Cil and/or Dex is complexed to the H-Dot and molar substitution ratios determined using ultraviolet (UV) spectrophotometry. Details of each step in the synthesis and complexation of nanotherapeutic components are provided in Supplementary Methods. The 3-dimensional structure of β-cyclodextrin was selected as the docking receptor (Protein Data Bank ID code: 1BFN). The ligand was energy minimized with the Chemistry at HARvard Macromolecular Mechanics force field, and the model was selected with the lowest binding energy using UCSF Chimera 1.15. The drug release tests of Cil/H-Dot and Dex/H-Dot complexes were conducted using rapid equilibrium dialysis devices (8-kDa molecular weight cutoff, Thermo Scientific). Research designated human donor kidneys for primary culture of PTECs (hPTECs) were provided by the Pacific Northwest Transplant Bank. The renal cortex was dissected, minced, and digested, followed by culture and passage as described.23Wallace D.P. Reif G.A. Generation of primary cells from ADPKD and normal human kidneys.Methods Cell Biol. 2019; 153: 1-23Crossref PubMed Scopus (4) Google Scholar CD10+/CD13+ live cells were selected by fluorescence-activated cell sorting as hPTEC for following experiments. A list of reagents for hPTEC is provided in Supplementary Table S1. A list of antibodies for fluorescence-activated cell sorting is provided in Supplementary Table S2. Mouse kidneys were perfusion fixed, embedded, and sectioned, followed by immunostaining for kidney injury molecule-1 (KIM-1), early endosomal antigen-1 (EEA-1), or megalin. For immunofluorescence in vitro, hPTEC were cultured on collagen-coated coverslips. A list of reagents and antibodies used for immunofluorescence is provided in Supplementary Table S2. Cil/fluorescein isothiocyanate (FITC)-Dex/H-Dot was administered to hPTEC (3.0 μM, 6 hours) grown on collagen-treated glass coverslips. High-power images were analyzed in CellProfiler software24Stirling D.R. Swain-Bowden M.J. Lucas A.M. et al.CellProfiler 4: improvements in speed, utility and usability.BMC Bioinformatics. 2021; 22: 433Crossref PubMed Scopus (529) Google Scholar (see Supplementary File). Puncta, nucleus, and cytoplasm were segmented and measured. To assess endosomal H-Dot component distribution, for each puncta, maximum Feret diameter and normalized intensity at 800 and 530 nM were computed. In vivo experiments were conducted on male mice as approved by Institutional Animal Care and Use committees at the University of Massachusetts Lowell, Massachusetts General Hospital, and Portland Veterans Affairs Medical Center. Each formulation (25 nmol) was i.v. injected at a 1000 nmol/kg dose into CD-1 mice 4 hours before imaging. Blood was collected and the plasma concentration of the H-Dot complexes quantified using the fluorescence signal. Pharmacokinetic analysis and modeling were performed by noncompartmental analysis using MATLAB 2023a/SimBiology 6.3 (MathWorks). The Dex liver, kidney, and urine concentration was quantified using the enzyme-linked immunosorbent assay (Forensic ELISA Kit, Neogen). Male CD-1 mice were s.c. injected with native Dex (Dex without nanoparticle conjugation), Dex/H-Dot, Cil/Dex/H-Dot, or saline (serving as a vehicle control) and subjected to the tail suspension test25Cryan J.F. Mombereau C. Vassout A. The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice.Neurosci Biobehav Rev. 2005; 29: 571-625Crossref PubMed Scopus (1255) Google Scholar 4 hours postadministration, followed by euthanasia and tissue collection. To determine the effect of Dex on immunosuppression, blood was collected via submandibular bleeding before administration (0 hours) and 4 hours after administration. Glycerol injection into the hind limb of mice induces myocyte necrolysis and elevates plasma myoglobin, causing AKI.26Oken D.E. Arce M.L. Wilson D.R. Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of oliguria.J Clin Invest. 1966; 45: 724-735Crossref PubMed Google Scholar Based on prior model optimization,18Matsushita K. Mori K. Saritas T. et al.Cilastatin ameliorates rhabdomyolysis-induced AKI in mice.J Am Soc Nephrol. 2021; 32: 2579-2594Crossref PubMed Scopus (25) Google Scholar 8 ml/kg of 50% glycerol was injected into the bilateral hind limbs of C57BL/6 mice under general anesthesia. Bilateral renal ischemia-reperfusion injury (IRI) was performed in male C57BL/6 mice as previously described.27Wei Q. Dong Z. Mouse model of ischemic acute kidney injury: technical notes and tricks.Am J Physiol Renal Physiol. 2012; 303: F1487-F1494Crossref PubMed Scopus (238) Google Scholar Body temperature was maintained in the range of 36.5 °C to 37.5 °C. Using sterile technique, bilateral flank incisions were made and renal pedicles simultaneously occluded. Twenty-five minutes later, pedicles were unclamped for reperfusion and the wounds were closed. The Cil/Dex/H-Dot in vivo dose was calculated to deliver to the proximal tubule the molar equivalent of a dose of Dex previously shown to be efficacious in AKI5Kumar S. Allen D.A. Kieswich J.E. et al.Dexamethasone ameliorates renal ischemia-reperfusion injury.J Am Soc Nephrol. 2009; 20: 2412-2425Crossref PubMed Scopus (101) Google Scholar,8Jacob K.A. Leaf D.E. Dieleman J.M. et al.Intraoperative high-dose dexamethasone and severe AKI after cardiac surgery.J Am Soc Nephrol. 2015; 26: 2947-2951Crossref PubMed Scopus (74) Google Scholar (detailed dosing calculation is given in Supplementary Methods). For experiments using the IRI model, 50 nmol Cil/Dex/H-Dot, Cil/H-Dot, Dex, or H-Dot without targeting and therapeutic moieties (empty H-Dot, H-Dot) was injected, half retro-orbitally and half s.c. immediately after renal reperfusion. In the glycerol injection model, 50 nmol Cil/Dex/H-Dot or H-Dot was injected immediately after glycerol injection. Delivery of Cil/Dex/H-Dot to inducible proximal tubule–specific megalin deletion (iMegKO) mice is described in Supplementary Methods. Twenty-four hours after IRI or glycerol injection, glomerular filtration rate (GFR) was quantified, followed by euthanasia and perfusion/fixation. GFR was measured using FITC-sinistrin as previously described.18Matsushita K. Mori K. Saritas T. et al.Cilastatin ameliorates rhabdomyolysis-induced AKI in mice.J Am Soc Nephrol. 2021; 32: 2579-2594Crossref PubMed Scopus (25) Google Scholar,28Matsushita K. Saritas T. Eiwaz M.B. et al.The acute kidney injury to chronic kidney disease transition in a mouse model of acute cardiorenal syndrome emphasizes the role of inflammation.Kidney Int. 2020; 97: 95-105Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar KIM-1 expression was quantified from immunofluorescence as % area. At 70% to 80% confluence, cells were transfected with small, interfering RNA directed at nuclear glucocorticoid receptor 3c1 or control small, interfering RNA. After 24 hours of transfection, cells were treated with Cil/Dex/H-Dot for 24 hours. Small, interfering RNAs used in the experiment are listed in Supplementary Table S3. After treatment with Cil/Dex/H-Dot, cell survival was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell survival was quantified as a percentage of controls. RNA was extracted from harvested cells and then reverse transcribed. Total RNA concentration was determined. TaqMan probes (listed in Supplementary Table S4) were used to amplify targets in <41 cycles of polymerase chain reaction and mRNA quantified in duplicate relative to glyceraldehyde-3-phosphate dehydrogenase by using the ΔΔCT method. Statistical analysis was performed in R (see Code Supplement) and Prism 9.4. Two-group comparisons were done using the t test or Mann-Whitney test, if non-Gaussian. Multiple group and time-repeated comparisons were done using analysis of variance with Sĭdák post-tests, unless there were missing data points, in which case linear mixed methods with a post hoc pairwise comparison was performed. Results are presented as mean ± SEM except where specifically noted. The nanotherapeutic H-Dot is composed of several key components including (i) a charge-balanced EPL backbone that enables renal clearance while preventing nonspecific binding; (ii) the zwitterionic NIR fluorophore ZW800, conjugated to ε-poly-l-lysine to allow for visualization and monitoring of the nanoparticles; and (iii) drug carriers consisting of β-cyclodextrin to facilitate multivalent drug delivery.14Kang H. Gravier J. Bao K. et al.Renal clearable organic nanocarriers for bioimaging and drug delivery.Adv Mater. 2016; 28: 8162-8168Crossref PubMed Scopus (113) Google Scholar,29Yin X. Cui Y. Kim R.S. et al.Image-guided drug delivery of nanotheranostics for targeted lung cancer therapy.Theranostics. 2022; 12: 4147-4162Crossref PubMed Google Scholar 1H nuclear magnetic resonance spectroscopy demonstrated that an average of 8 CD moieties had been grafted onto the ε-poly-l-lysine backbone (Supplementary Figure S1). To assess the surface charge of the H-Dot nanoparticles, we used both the ninhydrin test and UV-visible light-NIR spectroscopy. These methods revealed that ∼51% of the primary amines (positive charges) were converted into carboxylates (negative charges), resulting in a nearly zwitterionic surface on the final H-Dot (Supplementary Figure S2). In the following step, Cil and Dex were payloaded separately in the CD cavity of H-Dots for targeting megalin-bearing epithelial cells within the kidney and treatment of AKI, respectively (Figure 1). The inclusion complexation of H-Dots with Cil and Dex was revealed by docking simulation. This simulation indicated that the binding energies of Cil and Dex with CDs were −4.7 and −5.3 kcal/mol, respectively (Figure 2a). This finding suggests that the inclusion complexes are highly stable under physiological conditions. Cil and Dex were loaded into H-Dot nanoparticles either separately or together (Cil/H-Dot, Dex/H-Dot, and Cil/Dex/H-Dot). The molar ratios of Cil and Dex to H-Dot were calculated using UV-visible light-NIR spectroscopy and found to be 1.2 and 2.3, respectively (Figure 2b and c). After preparing inclusion complexes, the optical properties of H-Dots were measured using UV-visible light-NIR spectroscopy. The absorption and fluorescence emission peaks for the kidney-targeted AKI therapeutic nanoparticles were found to have maximum wavelengths of 785 and 801 nm, respectively (Figure 2c). Because a renally filtered drug is exposed to low urine pH in addition to physiologic pH, we further evaluated drug release potential of Dex from H-Dots at pH 5.0 by measuring changes in UV absorption spectra (Figure 2d). Approximately 70% of Cil and Dex was released from the H-Dot complexes after 8 hours of incubation and >80% after 24 hours of incubation. These findings suggest that Cil and Dex are effectively released from the H-Dot complex upon delivery to an acidic environment. H-Dots have been evaluated in drug delivery for various tumors,14Kang H. Gravier J. Bao K. et al.Renal clearable organic nanocarriers for bioimaging and drug delivery.Adv Mater. 2016; 28: 8162-8168Crossref PubMed Scopus (113) Google Scholar,15Kang H. Han M. Xue J. et al.Renal clearable nanochelators for iron overload therapy.Nat Commun. 2019; 10: 5134Crossref PubMed Scopus (98) Google Scholar,29Yin X. Cui Y. Kim R.S. et al.Image-guided drug delivery of nanotheranostics for targeted lung cancer therapy.Theranostics. 2022; 12: 4147-4162Crossref PubMed Google Scholar where the unbound molecules and delivery vehicles show rapid renal clearance with minimum to none off-target uptake. To this end, we evaluated the potential of H-Dots for kidney-targeted delivery of Cil and Dex to treat AKI without the adverse effects (Figure 3). First, the biodistribution of Cil/H-Dot, Dex/H-Dot, and Cil/Dex/H-Dot was investigated in CD-1 mice. The biodistribution pattern of the Dex/H-Dot complex was found to be similar to that of H-Dot itself in all organs with a weak NIR fluorescent signal in the kidney. Notably, a strong fluorescence signal was found in the bladder because of the rapid renal clearance of the H-Dot moiety. In the case of Cil/H-Dot and Cil/Dex/H-Dot complexes, much stronger signals were observed in the kidneys compared to control H-Dot. In addition, real-time imaging confirmed the excellent renal clearance of Cil/Dex/H-Dot (Figure 3a and b), and the signal-to-background ratio of the kidney after Cil/Dex/H-Dot administration was ∼2-fold higher than that of any other organs (Supplementary Figure S3). These results suggested that Cil in Cil/H-Dot and Cil/Dex/H-Dot has targeting potential for megalin-containing epithelial cells in the kidney. Because studies inconsistently suggest that some AKI models may reduce renal megalin levels,30Nielsen R. Christensen E.I. Birn H. Megalin and cubilin in proximal tubule protein reabsorption: from experimental models to human disease.Kidney Int. 2016; 89: 58-67Abstract Full Text Full Text PDF PubMed Google Scholar we performed pharmacokinetic modeling, varying GFR and proximal tubule megalin. These studies demonstrated that loss of renal megalin minimally delays but does not reduce Dex delivery to the proximal tubule (Supplementary Figure S4). To characterize the pharmacokinetics of therapeutic nanoparticles, we i.v. administered H-Dot to male CD-1 mice (1000 nmol/kg) and measured plasma concentrations of nanoparticles over time. All 3 nanoparticles exhibited plasma concentrations that disappeared biexponentially (Figure 4a). Interestingly, administration of Cil/Dex/H-Dot and Dex/H-Dot demonstrated generally lower plasma concentrations compared with H-Dot alone. These results were consistent with increased elimination rate constant (kel), shorter half-life (T1/2), decreased mean residence time, lower area under the plasma concentration-time curve, and higher clearance of Cil/Dex/H-Dot or Dex/H-Dot compared to H-Dot alone (Table 1). In addition, the urinary excretion of nanoparticles was increased after administration of Cil/Dex/H-Dot and Dex/H-Dot compared to H-Dot alone, which is congruent with the rapid plasma clearance of Cil/Dex/H-Dot and Dex/H-Dot. Cil/Dex/H-Dot displayed a decreased amount of nanoparticle recovered in urine (75% of the administered dose) compared with Dex/H-Dot (94%), suggesting that the addition of Cil could increase the residence time of H-Dot in the kidney, likely by facilitating megalin-mediated uptake of H-Dot into tubular epithelial cells. To evaluate the potential risk of off-target delivery and the benefit of kidney tissue delivery, compared to those of native Dex, we assessed the kidney:liver and kidney:urine ratios of Dex 4 hours after injection of native Dex, Dex/H-Dot, or Cil/Dex/H-Dot (Figure 4b). Complexation of Dex with the H-Dot nucleus likely improves kidney:liver specificity over native Dex, with addition of Cil likely improving kidney tissue retention of Dex. Taken together, these data characterize the novel pharmacokinetics of Cil/Dex/H-Dot.Table 1Pharmacokinetic parameters of H-Dot after a single i.v. injection to miceParameterParameter descriptionH-DotDex/H-DotCil/Dex/H-DotDose, nmol/kgInjected dose per kilogram of body weight100010001000kel, min−1Total body elimination rate constant0.0194 ± 0.00520.0279 ± 0.00490.0356 ± 0.0231T1/2, minHalf-life35.81 ± 9.6824.81 ± 4.3419.48 ± 12.65MRT, minMean residence time39.69 ± 11.6729.68 ± 3.4826.81 ± 10.80Ct=0, nmol/mlEstimated plasma concentration at time 047.01 ± 10.4818.04 ± 2.6314.39 ± 6.71AUC, nmol/ml∗minArea under the plasma concentration-time curve453.08 ± 79.93256.56 ± 108.96251.98 ± 155.36CL, ml/min/kgTotal body clearance2.30 ± 0.393.95 ± 1.724.20 ± 2.80VSS, ml/kgVolume of distribution at the steady state91.35 ± 26.86117.15 ± 13.73112.48 ± 45.32AR, %% Dose amount excreted via urine63 ± 1594 ± 375 ± 15Cil, cilastatin; Dex, dexamethasone.Data are expressed as mean ± SD. Open table in a new tab Cil, cilastatin; Dex, dexame