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Accumulation and cellular localization of nanoparticles in an ex vivo model of acute lung injury
j o u r n a l o f s u r g i c a l r e s e a r c h a p r i l 2 0 1 7 ( 2 1 0 ) 7 8 e8 5
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Accumulation and cellular localization of nanoparticles in an ex vivo model of acute lung injury Joshua C. Grimm, MD,a,1 Fan Zhang, PhD,b,c,1 Jonathan T. Magruder, MD,a Todd C. Crawford, MD,a Manoj Mishra, PhD,b Kannan M. Rangaramanujam, PhD,b,** and Ashish S. Shah, MDd,* a
Division of Cardiac Surgery, Department of Surgery, The Johns Hopkins Medical Institution, Baltimore, Maryland Department of Ophthalmology, Center for Nanomedicine, The Johns Hopkins Medical Institution, Baltimore, Maryland c Department of Material Sciences and Engineering, The Johns Hopkins Medical Institution, Baltimore, Maryland d Department of Cardiac Surgery, Vanderbilt University Medical Center, Nashville, Tennessee b
article info
abstract
Article history:
Background: The benefit of nanomedicine in mitigating acute lung injury (ALI) is currently
Received 25 August 2016
unknown. Therefore, we introduced the generation IV polyamidoamine dendrimers with
Received in revised form
neutral surface property (dendrimer) into our established ex vivo animal model and sought to
18 October 2016
determine their biodistribution to define their cellular uptake profile and to evaluate their
Accepted 2 November 2016
potential as a drug delivery candidate for the treatment of ischemiaereperfusioneinduced ALI.
Available online 9 November 2016
Methods: Eight rabbit heartelung blocks were harvested and exposed to 18 h of cold ischemia. The heartelung blocks were then reperfused with rabbit donor blood. Dendrimer
Keywords:
was conjugated to fluorescein isothiocyanate (D-FITC) for localization and quantification
Nanoparticle therapy
studies. D-FITC (30 mg or 150 mg) was injected into the bypass circuit and baseline, 1- and
Acute lung injury
2-h tissue samples were obtained to determine percent uptake. Low (10) and high (40)
Animal model
magnification images were obtained using confocal microscopy to confirm the accumulation and to determine the cellular targets of the dendrimer. Results: Four heartelung blocks were exposed to 30 mg and four to 150 mg of D-FITC. After adjusting for dry weight, the mean uptake in the 30 and 150 mg samples after 2 h of reperfusion were 0.79 0.16% and 0.39 0.22% of perfused doses, respectively. Confocal imaging demonstrated dendrimer uptake in epithelial cells and macrophages. Conclusions: Fluorescently tagged dendrimers demonstrated injury-dependent tissue accumulation in a variety of different cell types. This unique approach will allow conjugation to and delivery of multiple agents with the potential of mitigating ALI injury while avoiding systemic toxicity. ª 2016 Elsevier Inc. All rights reserved.
The results of this study were presented at the 2014 ISHLT Annual Meeting and Scientific Sessions. * Corresponding author. Department of Cardiac Surgery, Vanderbilt University Medical Center, 1215 21st Avenue South, Suite 5025, Nashville, TN 37232. Tel.: þ1 (615) 343-7363; fax: þ1 (443) 287-4226. ** Corresponding author. Department of Ophthalmology, Center for Nanomedicine, Johns Hopkins School of Medicine, 400 North Broadway, Smith 6023, Baltimore, MD 21287. Tel.: þ1 443-287-8634; fax: þ1 443-287-8635. E-mail addresses: [email protected] (K.M. Rangaramanujam), [email protected] (A.S. Shah). 1 Co-first authors. 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.11.007
grimm et al accumulation and cellular localization of nanoparticles in an ex vivo model of ali
Introduction
Materials and methods
Acute lung injury (ALI) has a considerable impact on survival and quality life.1,2 Because of inconsistent definitions and institutional underreporting, the exact incidence of ALI is unknown; but, based on the results of several large, observational studies, it appears to range between 65 and 90 cases per 100,000 persons.1,3,4 ALI can occur after a direct pulmonary insult, namely pneumonia or lung transplantation, or, secondarily, as a result of a number of extra-pulmonary processes, such as trauma, massive transfusion, and abdominal catastrophe.5,6 Clinical manifestations and diagnostic criteria include hypoxemia and bilateral infiltrates in the context of normal cardiac filling pressures.7 The typical histologic triad of neutrophilic alveolitis, hyaline membrane deposition, and microthrombi formation gives further insight into its pathogenesis and pattern of disease progression. Given its deleterious effects on outcomes, research continues into prophylactic measures and novel therapies aimed at retarding its development and combating its clinical influence. To promote a further understanding into the inflammatory milieu associated with ALI, various animal platforms have been used to recreate an injury profile consistent with the aforementioned clinical and histologic findings. Although each has explicit limitations, ischemiaereperfusion injury (IRI) appears to reliably initiate a cascade of cellular events, which culminates in endothelial leak and necrosis.8,9 Thus, the IRI model can be used in the evaluation of therapeutic interventions for ALI. Current approaches in drug development to address this devastating disorder are greatly hindered by poor pharmacokinetic profiles and the nonselective nature of these treatments against the diverse mechanisms underlying the pathogenesis of ALI.4 Nanomedicine-based delivery platforms may mitigate these challenges by altering drug biodistribution profiles and targeting the major effectors in the inflammatory cascade. The microvascular permeability associated with ALI allows for the extravasation of systemically administrated nanoparticles to the focus of injury.4 In addition, the selective cell-targeting property of nanoparticles may allow for the identification of specific pathway intermediaries for therapeutic alteration.10 Our group has previously used generation IV polyamidoamine (PAMAM) dendrimers with “neutral” hydroxyl surface functionalities to successfully deliver drugs to activated microglia/macrophages11-14 producing neurologic improvements in animal models of cerebral palsy, hypothermic circulatory arrest, and ischemia.12-14 Some previous studies using dendrimers have focused on the feasibility of direct pulmonary15 or gastrointestinal16 absorption of dendrimer nanoparticles to mitigate lung inflammation. There are clinical limitations in the gastrointestinal administration of medications given the variability of absorption and metabolism, especially in patients with acute illness requiring hemodynamic support. Accordingly, we introduced fluorescein-conjugated dendrimers into the bypass circuit of our established ex vivo model to determine its biokinetics after systemic administration as well as to investigate the influence of IRI on dendrimer tissue accumulation after 1 and 2 h of reperfusion.
Preparation of dendrimer-fluorescein isothiocyanate conjugate
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The dendrimer-fluorescein isothiocyanate (FITC) conjugates were prepared in two steps by following an established method with a small modification.17 In brief, the surface of hydroxyl terminated generation 4.0 PAMAM dendrimers (G4-OH; Dendritech, Midland, MI) was partially modified with amine groups to form a bifunctional dendrimer. First, 6-(Fmoc-amino)caproic acid was conjugated to G4-OH, using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate as a coupling reagent and N,N-diisopropylethylamine as base. The Fmoc-protected intermediate was then deprotected with a piperidine and dimethylformamide mixed solution (piperidine:dimethylformamide ¼ 1:9) to form the bifunctional dendrimer. In the next step, FITC reacted with the bifunctional dendrimer using a borate buffer (pH ¼ 8.5). The reaction mixture was then purified by dialysis against deionized water. The final D-FITC conjugate was characterized by proton nuclear magnetic resonance, high-performance liquid chromatography, and gel permeation chromatography.
Procurement of the heartelung blocks The surgical technique of procuring rabbit heartelungs for manipulation on an ex vivo circuit has been described, but a brief narrative is included. This surgical prep has been validated in several studies as an excellent model of ALI.18 A total of three 3-5 kg New Zealand white rabbits (Robinson Services, Inc, Mocksville, NC) are used for each experiment. One rabbit provides the heartelung block to be cannulated and ventilated on the ex vivo platform, whereas two others function as blood donors for the cardiopulmonary bypass circuit. After appropriate anesthesia is achieved with a combination of intramuscular ketamine (35 mg/kg) and xylazine (6.5 mg/kg) and intravenous acepromazine (5 mg/kg), a cut down tracheostomy is performed and median sternotomy incision is used to gain access to the thoracic cavity. The rabbit is ventilated on a volume control mode at a rate of 20 breaths per minute, a tidal volume of 10 mL/kg, and a fraction of inspired concentration of oxygen of 100% (Harvard ventilator apparatus, model 665; Harvard Apparatus, Holliston, MA). The superior vena cava, inferior vena cava, pulmonary artery, and aorta are identified and encircled with suture. Intracardiac prostaglandin (30 mg) and a bolus of intravenous heparin (1000 U/kg) are administered. A right ventriculotomy is fashioned, and an inflow cannula is advanced into the main pulmonary artery. An outflow cannula is then positioned in the left atrium and secured. The inferior vena cava, superior vena cava, and aorta are then ligated, instillation of 250 mL of cold (4 C) Perfadex (XVIVO, Goteborg, Sweden) via the inflow cannula is initiated and the heartelung block is dissected from the chest cavity (Fig. 1). All the animals in this experiment were treated in a humane fashion in accordance with our institutional animal care and use guidelines.
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Reperfusion of heartelung blocks After heparinization, two rabbits undergo exsanguination via a right ventricular cannula to provide 300 cc of blood for the bypass circuit. A left atrial cannula is introduced into the heartelung block and secured for blood pressure monitoring. The rabbit lung is ventilated with the aforementioned settings and the Sarns 5000 roller head pump cardiopulmonary bypass circuit (Sarns, Ann Arbor, MI) is de-aired.
Lung samples Lung tissue is removed at the following time points throughout the course of the experiment1: 1 h after the initiation of reperfusion and2 at the completion of the experiment (2-h reperfusion). Tissue is either frozen with liquid nitrogen and stored at 80 C for quantification or preserved in 4% paraformaldehyde for confocal imaging.
Determining the influence of dose, reperfusion duration, and injury status on D-FITC accumulation To determine the influence of the D-FITC reperfusion dose on lung uptake after reperfusion, two doses (30 or 150 mg) were bolused into the blood perfusate and allowed to continuously
circulate for the duration of the 2-h experiment. D-FITC tissue levels were then assessed at time point2 to determine the ultimate percent uptake. Based on this information, a D-FITC reperfusion dose was identified for subsequent parts of the experiment. To compare the uptake of dendrimer in injured and uninjured tissue after procurement, one set of heartelung blocks is cold stored (4 C) in Perfadex solution for 18 h (ischemic insult). A bolus of the D-FITC is then administered into the bypass circuit and allowed to continuously circulate for the duration of the 2-h experiment with tissue removed after 1- and 2-h reperfusion. Lungs randomized to the uninjured cohort underwent left atrial cannulation and ventilation and reperfusion immediately after the procurement without a cold-ischemic insult. The perfusate was bolused in a similar manner with D-FITC, and tissue was removed as outlined.
Quantification of D-FITC A novel fluorescence-based quantification method with high sensitivity (reaching 10 ng levels of detection) was used to measure dendrimer accumulation in the lung tissue removed at time points 1 and 2.19 Approximately 100 mg of lung tissue was homogenized in 1 mL of methanol in 2-mL DNA LoBind Eppendorf tubes (TissueLyser LT; Qiagen, Hilden, Germany). After homogenization, no intact cellular structures were observed under the microscope. Tissue homogenates were then diluted to 100 mg/mL and sonicated for 15 min to fully extract the D-FITC from the specimen. The resulting homogenates were centrifuged (15,000 rpm) at 4 C for 15 min to separate the supernatants from the tissue pellet. The fluorescence spectrum of the resulting supernatants was measured using a Shimadzu RF-5301 Spectrofluorophotometer (Shimadzu, Japan). D-FITC in each sample was quantified by comparing the measured fluorescence (lext ¼ 495 nm and lemi ¼ 520 nm), against an appropriate calibration curve. The ultimate tissue quantity was calculated by constructing a D-FITCebased calibration curve (R2 ¼ 0.98) of the recorded intensities (expressed as percentage of injected dose per gram of tissue [%ID/g]).
Immunofluorescence study of dendrimer distribution
Fig. 1 e Heartelung block on ex vivo circuit. The pulmonary artery cannula provides inflow (black-filled arrow) and the left atrial cannula outflow (white-filled arrow) on the cardiopulmonary bypass circuit. Tracheal intubation noted with white asterisk (*). (Color version of figure is available online.)
Procured tissues were fixed with 4% paraformaldehyde immediately after harvesting, sequentially processed with increasing concentrations of a sucrose solution (10%-30%) and cryosectioned. Tissues were sectioned transversely into 10mm thick slices using a Leica CM 1905 cryostat. These slices were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Life technology, Frederick, MD) and Tomatolectin-Texas Red (Vector Labs, Burlingame, CA). The samples were then imaged using a confocal LSM 710 microscope (Carl Zeiss, Hertfordshire, UK) under 20 and 40 magnification. For each image, settings were carefully optimized based on noneD-FITC perfused rabbit lung controls to avoid background fluorescence. Laser power, pinhole, gain, offset, and digital gain were selected separately for each magnification and kept consistent throughout the entire study.
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Fig. 2 e D-FITC accumulation in the ischemiaereperfusion lung represented in microgram (A) and as a percentage of the reperfusion dose (B). The weight of two rabbit lungs was 20 g. The low and high bolus doses were 30 and 150 mg, respectively. Two additional rabbits were needed for the 2-h, low bolus group to augment tissue quantity for the assay.
Results Dendrimer-FITC lung accumulation as a function of dose, time, and degree of injury To gain a better understanding of dendrimer uptake kinetics in this ex vivo model, we quantified D-FITC accumulation at different bolus doses, reperfusion durations, and injury status. Four experiments were performed to determine uptake after 1 h of reperfusion in both the low and the high bolus dose groups. An additional two experiments were performed in the
low bolus dose group to obtain enough sample for quantification. At the lower reperfusion dose (30 mg), 187-236 mg of D-FITC accumulated in the lung tissue, corresponding to 0.63%-0.79% of the reperfusion dose. When the initial reperfusion dose increased fivefold (150 mg), we found an increase of D-FITC accumulation to 343-586 mg (twofold increase). This demonstrates a decrease in the accumulation of D-FITC in relation to the perfused dose, indicating a possible ceiling for D-FITC uptake by the damaged lung (Fig. 2). Based on these findings, we identified a D-FITC reperfusion dose of 90 mg to investigate the effects of reperfusion time and injury status on D-FITC accumulation. Four experiments were performed in each arm of this portion of the study (both the control and experimental groups). Our findings demonstrate greater D-FITC uptake by injured lung tissue after 2 h of reperfusion. Specifically, after 1 h of reperfusion, both the injured and uninjured lungs exhibited similar D-FITC uptake (w0.20% of the perfused dose). After 2 h, however, D-FITC accumulation in injured lung specimen increased 100% (w0.40% of reperfusion dose), whereas remaining relatively unchanged in the uninjured lungs (w0.21%; Fig. 3).
Dendrimers target pneumocytes and alveoari macrophages
Fig. 3 e D-FITC accumulation in the control and injured lungs as a function of time. The weight of two rabbit lungs was 20 g. The bolus dose used in this experiment was 90 mg dendrimer per lung. The sample size for each group is 4.
As stated previously, one of the proposed therapeutic benefits of dendrimer-based delivery is its ability to selectively target the injured cells. Accordingly, we used immunofluorescence staining and confocal microscopy to determine the ultimate cellular distribution of the D-FITC. Interestingly, D-FITC was identified in the major histologic subtypes (epithelium and inflammatory) by the completion of reperfusion (Fig. 4). Based on anatomy and the cell morphology, we showed that, compared with the tissue background (Fig. 4A), the D-FITC colocalized with the pneumocytes in the alveoli (Fig. 4B) and bronchiole (Fig. 4C) from the injured lung tissue. In addition, we found some uptake of D-FITC by alveoli macrophages (Fig. 4D). Expectantly, the 1-h samples demonstrated a limited amount of D-FITC uptake at the cellular level. After 2 h of
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Fig. 4 e Confocal imaging of D-FITC distribution in the ischemiaereperfusion lung demonstrating targeted cell uptake. (A) Section without dendrimer reperfusion; (B) D-FITC accumulation in the pneumocytes; (C) D-FITC accumulation in the bronchiole; (D) D-FITC accumulation in the alveolar macrophages. The white arrow indicates dendrimer accumulation. The scale bar in the image is 20 mm. I: Alveoli; II: Pneumocytes; III: Bronchiole; IV: Alveoli macrophage. Red: Lectin, Green: D-FITC, Blue: DAPI. (Color version of figure is available online.)
reperfusion, however, D-FITC was identified in all cell types (Fig. 5).
Discussion The feasibility of intravenously introducing nanoparticles into an ex vivo model of ALI was previously unknown. We demonstrated that D-FITC is not only preferentially taken up by damaged lung tissue but that it also shows a timedependent accumulation in specific cellular targets. The latter could have profound implications on future dendrimerdrug conjugateebased therapies aimed at minimizing the morbidity and mortality of ALI. The pathogenesis of this inflammatory process is critically important when devising a successful delivery platform. The mechanism involves infiltration of the lung tissue by polymorphonuclear neutrophils whose local activation results in subsequent injury.7 As pro-inflammatory cytokines are
released, a cascade of events is initiated and ultimately leads to endothelial damage and increased capillary permeability.20 Previous studies on capillary permeability in ALI indicated variations in the degree of capillary damage based on the infiltration coefficient,21,22 which might hinder the extravasation of nanoparticles of a larger size at the site of injury. The small size (w4 nm in diameter) and nearly neutral surface charge (z-potential: þ4.5 0.1 mV) of the PAMAM dendrimer constructed for these experiments undoubtedly contributed to its ability to traverse the endothelium and gain access to its cellular targets.23 As prolonged reperfusion resulted in a greater severity of lung injury and further deterioration in cellto-cell adhesion, an expected increase in D-FITC accumulation was noted. This was not only true in the proximal airway and segmental bronchi, but also at the anatomic level of pulmonary gas exchange. Given the injury profile associated with ALI, dendrimerdrug conjugate nanotherapy could prove efficacious in affecting the major cellular components of repair while
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Fig. 5 e Confocal imaging demonstrating dendrimer accumulation in lung tissue before reperfusion and after 1 and 2 h of reperfusion. The area shown in the image was taken under 203 with tilescan and the sample size is approximately to 1 3 1 mm. The scale bar in the image is 100 mm. The white arrow indicates dendrimer accumulation. Red: Lectin, Green: DFITC, Blue: DAPI. (Color version of figure is available online.)
simultaneously minimizing systemic toxicity. Dendrimer nanoparticles have been previously shown to localize to damaged areas of the lung.16 More specifically, an investigation into direct pulmonary administration (via inhalation) of a PAMAM dendrimer-methylprednisolone conjugate for the treatment of asthma demonstrated that conjugated therapy resulted in a significant decrease in the concentration of inflammatory cells in comparison to free drug alone.15 There are several limitations, however, in extrapolating these findings, and assuming similar efficacy, in the treatment of ALI. First, and perhaps most importantly, the structural changes and inflammatory response associated with asthma are different in regards to their magnitude, duration, and cellular effectors than that experienced in ALI. In the latter, gross pulmonary edema most likely limits the biodistribution of inhaled dendrimer in the distal airway. Another study established the beneficial role of dendrimer therapy in mitigating the severe lung injuryeinflammation after LPS endotoxemia.16 Again, however, the gastrointestinal route of drug administration could limit the clinically applicability of this treatment in patients with extreme cases of ALI. Neither of the aforementioned studies explored the specific cellular localization of the dendrimer.
Although these prior investigations have demonstrated some success in ameliorating lung injury in animal models, not only can the route of delivery be clinically problematic but there is also a lack of understanding of the actual pathways involved in the anti-inflammatory effects. The present study represents the first known demonstration of the cellular-level localization of dendrimer in lung tissue following systemic administration. As stated, the dendrimer was identified in the major alveolar cell subtypes (epithelial and inflammatory). This extends the possibility of using dendrimer-based, conjugated therapies in multiple capacities in the inflammatory cascade of ALI. This should not be underemphasized as it is known that pneumocytes (epithelial cells) undergo shedding and alterations in function as a result of ALI.24 Previous studies have demonstrated that this pattern of injury in type II pneumocytes results in the production of dysfunctional surfactant, a phospholipid instrumental in maintaining normal alveolar surface tension.25,26 And, whereas previous attempts at exogenous surfactant administration in the treatment of ALI have yield inconsistent results, dendrimer conjugates could improve bioavailability and offer additional routes for drug administration.27-29 Similar strategies could be used to target circulating macrophages, which have not only been
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implicated in the development of ALI30,31 but also its resolution.32
references
Limitations
1. Rubenfeld GD, Caldwell E, Peabody E, et al. Incidence and outcomes of acute lung injury. N Engl J Med. 2005;353:1685e1693. 2. Dowdy DW, Eid MP, Dennison CR, et al. Quality of life after acute respiratory distress syndrome: a meta-analysis. Intensive Care Med. 2006;32:1115e1124. 3. Goss CH, Brower RG, Hudson LD, Rubenfeld GD. Incidence of acute lung injury in the United States. Crit Care Med. 2003;31:1607e1611. 4. Sadikot RT. Peptide nanomedicines for treatment of acute lung injury. Methods Enzymol. 2012;508:315e324. 5. Stoclin A, Delbos F, Dauriat G, et al. Transfusion-related acute lung injury after intravenous immunoglobulin treatment in a lung transplant recipient. Vox Sang. 2013;104:175e178. 6. Fremont RD, Koyama T, Calfee CS, et al. Acute lung injury in patients with traumatic injuries: utility of a panel of biomarkers for diagnosis and pathogenesis. J Trauma. 2010;68:1121e1127. 7. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;342:1334e1349. 8. Neely CF, Keith IM. A1 adenosine receptor antagonists block ischemia-reperfusion injury of the lung. Am J Physiol. 1995;268:L1036eL1046. 9. Sakao Y, Kajikawa O, Martin TR, et al. Association of IL-8 and MCP-1 with the development of reexpansion pulmonary edema in rabbits. Ann Thorac Surg. 2001;71:1825e1832. 10. Hayder M, Poupot M, Baron M, et al. A phosphorus-based dendrimer targets inflammation and osteoclastogenesis in experimental arthritis. Sci Transl Med. 2011;3:81ra35. 11. Zhang F, Nance E, Alnasser Y, Kannan R, Kannan S. Microglial migration and interactions with dendrimer nanoparticles are altered in the presence of neuroinflammation. J Neuroinflammation. 2016;13:65. 12. Mishra MK, Beaty CA, Lesniak WG, et al. Dendrimer brain uptake and targeted therapy for brain injury in a large animal model of hypothermic circulatory arrest. ACS Nano. 2014;8:2134e2147. 13. Kannan S, Dai H, Navath RS, et al. Dendrimer-based postnatal therapy for neuroinflammation and cerebral palsy in a rabbit model. Sci Transl Med. 2012;4:130ra46. 14. Grimm JC, Magruder JT, Wilson MA, et al. Nanotechnology approaches to targeting inflammation and excitotoxicity in a canine model of hypothermic circulatory arrest-induced brain injury. Ann Thorac Surg. 2016;102:743e750. 15. Inapagolla R, Guru BR, Kurtoglu YE, et al. In vivo efficacy of dendrimer-methylprednisolone conjugate formulation for the treatment of lung inflammation. Int J Pharm. 2010;399:140e147. 16. Blattes E, Vercellone A, Eutamene H, et al. Mannodendrimers prevent acute lung inflammation by inhibiting neutrophil recruitment. Proc Natl Acad Sci U S A. 2013;110:8795e8800. 17. Mishra MK, Kotta K, Hali M, et al. PAMAM dendrimerazithromycin conjugate nanodevices for the treatment of Chlamydia trachomatis infections. Nanomedicine. 2011;7:935e944. 18. George TJ, Arnaoutakis GJ, Beaty CA, et al. Hydrogen sulfide decreases reactive oxygen in a model of lung transplantation. J Surg Res. 2012;178:494e501. 19. Lesniak WG, Mishra MK, Jyoti A, et al. Biodistribution of fluorescently labeled PAMAM dendrimers in neonatal rabbits: effect of neuroinflammation. Mol Pharm. 2013;10:4560e4571. 20. Zemans RL, Matthay MA. Bench-to-bedside review: the role of the alveolar epithelium in the resolution of pulmonary edema in acute lung injury. Crit Care. 2004;8:469e477.
In spite of the interesting cellular localization of the dendrimers, the findings of this experiment are based on a limited sample size. In addition, although IRI has been established as a reliable surrogate for ALI, we appreciate that subtle differences undoubtedly exist at the cellular level between these two processes. This could affect the degree of nanoparticle extravasation and targeted tissue uptake. Given that uptake in the control lungs was so trivial at the 1-h interval, and persisted at the 2-h interval, it was difficult to discern which cell types consistently took up the nanoparticle. Accordingly, it was not possible to describe differences in the cellular targets between these two arms of the experiment. Future studies are required to determine the systemic uptake profile of the dendrimer to minimize systemic toxicities in the other end organs that might sequester the nanoparticle.
Conclusion Although ALI confers substantial morbidity and mortality to a wide range of patient populations, current treatment options are limited. Nanoparticle-based therapy offers a promising new approach to target the known cellular effectors involved in both the inflammatory and reparative processes. To our knowledge, this is the first study to demonstrate the successful administration of intravenous dendrimer on an ex vivo IRI model of ALI, to identify targets of its uptake, and to quantify the influence of injury on tissue accumulation. Future studies into dendrimer-drug conjugates could have profound implications on the clinical management of this condition and warrant additional investigation.
Acknowledgment The authors would like to thank the Wilmer Core Module for Microscopy and Imaging for allowing us to use LSM710 confocal microscopy. The authors also want to acknowledge Melissa Jones and Jeffrey Braun for their attention to detail in all facets of this experiment, from intraoperative surgical technique and anesthesia to postoperative care. This work was funded by NIBIB R01EB018306 (R.K.). Authors’ contributions: J.C.G., T.C.C., J.T.M., and F.Z. did the data analysis; J.C.G., F.Z., K.N.R., and A.S.S. contributed in methodology; J.C.G. and F.Z. wrote the article; K.N.R. and A.S.S. approved the final version of the article.
Disclosures The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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21. Parker JC, Townsley MI. Evaluation of lung injury in rats and mice. Am J Physiol Lung Cell Mol Physiol. 2004;286:L231eL246. 22. Parker JC. Acute lung injury and pulmonary vascular permeability: use of transgenic models. Compr Physiol. 2011;1:835e882. 23. Iezzi R, Guru BR, Glybina IV, et al. Dendrimer-based targeted intravitreal therapy for sustained attenuation of neuroinflammation in retinal degeneration. Biomaterials. 2012;33:979e988. 24. Stanley MW, Henry-Stanley MJ, Gajl-Peczalska KJ, Bitterman PB. Hyperplasia of type II pneumocytes in acute lung injury. Cytologic findings of sequential bronchoalveolar lavage. Am J Clin Pathol. 1992;97:669e677. 25. Jobe AH, Ikegami M. Surfactant and acute lung injury. Proc Assoc Am Physicians. 1998;110:489e495. 26. Veldhuizen RA, McCaig LA, Akino T, Lewis JF. Pulmonary surfactant subfractions in patients with the acute respiratory distress syndrome. Am J Respir Crit Care Med. 1995;152(6 Pt 1):1867e1871. 27. Anzueto A, Baughman RP, Guntupalli KK, et al. Aerosolized surfactant in adults with sepsis-induced acute respiratory
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distress syndrome. Exosurf acute respiratory distress syndrome sepsis study group. N Engl J Med. 1996;334:1417e1421. Gregory TJ, Steinberg KP, Spragg R, et al. Bovine surfactant therapy for patients with acute respiratory distress syndrome. Am J Respir Crit Care Med. 1997;155:1309e1315. Davidson WJ, Dorscheid D, Spragg R, et al. Exogenous pulmonary surfactant for the treatment of adult patients with acute respiratory distress syndrome: results of a metaanalysis. Crit Care. 2006;10:R41. Kooguchi K, Hashimoto S, Kobayashi A, et al. Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia. Infect Immun. 1998;66:3164e3169. Marriott HM, Dockrell DH. The role of the macrophage in lung disease mediated by bacteria. Exp Lung Res. 2007;33:493e505. Johnston LK, Rims CR, Gill SE, McGuire JK, Manicone AM. Pulmonary macrophage subpopulations in the induction and resolution of acute lung injury. Am J Respir Cell Mol Biol. 2012;47:417e426.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Accumulation and cellular localization of nanoparticles in an ex vivo model of acute lung injury Joshua C. Grimm, MD,a,1 Fan Zhang, PhD,b,c,1 Jonathan T. Magruder, MD,a Todd C. Crawford, MD,a Manoj Mishra, PhD,b Kannan M. Rangaramanujam, PhD,b,** and Ashish S. Shah, MDd,* a
Division of Cardiac Surgery, Department of Surgery, The Johns Hopkins Medical Institution, Baltimore, Maryland Department of Ophthalmology, Center for Nanomedicine, The Johns Hopkins Medical Institution, Baltimore, Maryland c Department of Material Sciences and Engineering, The Johns Hopkins Medical Institution, Baltimore, Maryland d Department of Cardiac Surgery, Vanderbilt University Medical Center, Nashville, Tennessee b
article info
abstract
Article history:
Background: The benefit of nanomedicine in mitigating acute lung injury (ALI) is currently
Received 25 August 2016
unknown. Therefore, we introduced the generation IV polyamidoamine dendrimers with
Received in revised form
neutral surface property (dendrimer) into our established ex vivo animal model and sought to
18 October 2016
determine their biodistribution to define their cellular uptake profile and to evaluate their
Accepted 2 November 2016
potential as a drug delivery candidate for the treatment of ischemiaereperfusioneinduced ALI.
Available online 9 November 2016
Methods: Eight rabbit heartelung blocks were harvested and exposed to 18 h of cold ischemia. The heartelung blocks were then reperfused with rabbit donor blood. Dendrimer
Keywords:
was conjugated to fluorescein isothiocyanate (D-FITC) for localization and quantification
Nanoparticle therapy
studies. D-FITC (30 mg or 150 mg) was injected into the bypass circuit and baseline, 1- and
Acute lung injury
2-h tissue samples were obtained to determine percent uptake. Low (10) and high (40)
Animal model
magnification images were obtained using confocal microscopy to confirm the accumulation and to determine the cellular targets of the dendrimer. Results: Four heartelung blocks were exposed to 30 mg and four to 150 mg of D-FITC. After adjusting for dry weight, the mean uptake in the 30 and 150 mg samples after 2 h of reperfusion were 0.79 0.16% and 0.39 0.22% of perfused doses, respectively. Confocal imaging demonstrated dendrimer uptake in epithelial cells and macrophages. Conclusions: Fluorescently tagged dendrimers demonstrated injury-dependent tissue accumulation in a variety of different cell types. This unique approach will allow conjugation to and delivery of multiple agents with the potential of mitigating ALI injury while avoiding systemic toxicity. ª 2016 Elsevier Inc. All rights reserved.
The results of this study were presented at the 2014 ISHLT Annual Meeting and Scientific Sessions. * Corresponding author. Department of Cardiac Surgery, Vanderbilt University Medical Center, 1215 21st Avenue South, Suite 5025, Nashville, TN 37232. Tel.: þ1 (615) 343-7363; fax: þ1 (443) 287-4226. ** Corresponding author. Department of Ophthalmology, Center for Nanomedicine, Johns Hopkins School of Medicine, 400 North Broadway, Smith 6023, Baltimore, MD 21287. Tel.: þ1 443-287-8634; fax: þ1 443-287-8635. E-mail addresses: [email protected] (K.M. Rangaramanujam), [email protected] (A.S. Shah). 1 Co-first authors. 0022-4804/$ e see front matter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2016.11.007
grimm et al accumulation and cellular localization of nanoparticles in an ex vivo model of ali
Introduction
Materials and methods
Acute lung injury (ALI) has a considerable impact on survival and quality life.1,2 Because of inconsistent definitions and institutional underreporting, the exact incidence of ALI is unknown; but, based on the results of several large, observational studies, it appears to range between 65 and 90 cases per 100,000 persons.1,3,4 ALI can occur after a direct pulmonary insult, namely pneumonia or lung transplantation, or, secondarily, as a result of a number of extra-pulmonary processes, such as trauma, massive transfusion, and abdominal catastrophe.5,6 Clinical manifestations and diagnostic criteria include hypoxemia and bilateral infiltrates in the context of normal cardiac filling pressures.7 The typical histologic triad of neutrophilic alveolitis, hyaline membrane deposition, and microthrombi formation gives further insight into its pathogenesis and pattern of disease progression. Given its deleterious effects on outcomes, research continues into prophylactic measures and novel therapies aimed at retarding its development and combating its clinical influence. To promote a further understanding into the inflammatory milieu associated with ALI, various animal platforms have been used to recreate an injury profile consistent with the aforementioned clinical and histologic findings. Although each has explicit limitations, ischemiaereperfusion injury (IRI) appears to reliably initiate a cascade of cellular events, which culminates in endothelial leak and necrosis.8,9 Thus, the IRI model can be used in the evaluation of therapeutic interventions for ALI. Current approaches in drug development to address this devastating disorder are greatly hindered by poor pharmacokinetic profiles and the nonselective nature of these treatments against the diverse mechanisms underlying the pathogenesis of ALI.4 Nanomedicine-based delivery platforms may mitigate these challenges by altering drug biodistribution profiles and targeting the major effectors in the inflammatory cascade. The microvascular permeability associated with ALI allows for the extravasation of systemically administrated nanoparticles to the focus of injury.4 In addition, the selective cell-targeting property of nanoparticles may allow for the identification of specific pathway intermediaries for therapeutic alteration.10 Our group has previously used generation IV polyamidoamine (PAMAM) dendrimers with “neutral” hydroxyl surface functionalities to successfully deliver drugs to activated microglia/macrophages11-14 producing neurologic improvements in animal models of cerebral palsy, hypothermic circulatory arrest, and ischemia.12-14 Some previous studies using dendrimers have focused on the feasibility of direct pulmonary15 or gastrointestinal16 absorption of dendrimer nanoparticles to mitigate lung inflammation. There are clinical limitations in the gastrointestinal administration of medications given the variability of absorption and metabolism, especially in patients with acute illness requiring hemodynamic support. Accordingly, we introduced fluorescein-conjugated dendrimers into the bypass circuit of our established ex vivo model to determine its biokinetics after systemic administration as well as to investigate the influence of IRI on dendrimer tissue accumulation after 1 and 2 h of reperfusion.
Preparation of dendrimer-fluorescein isothiocyanate conjugate
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The dendrimer-fluorescein isothiocyanate (FITC) conjugates were prepared in two steps by following an established method with a small modification.17 In brief, the surface of hydroxyl terminated generation 4.0 PAMAM dendrimers (G4-OH; Dendritech, Midland, MI) was partially modified with amine groups to form a bifunctional dendrimer. First, 6-(Fmoc-amino)caproic acid was conjugated to G4-OH, using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate as a coupling reagent and N,N-diisopropylethylamine as base. The Fmoc-protected intermediate was then deprotected with a piperidine and dimethylformamide mixed solution (piperidine:dimethylformamide ¼ 1:9) to form the bifunctional dendrimer. In the next step, FITC reacted with the bifunctional dendrimer using a borate buffer (pH ¼ 8.5). The reaction mixture was then purified by dialysis against deionized water. The final D-FITC conjugate was characterized by proton nuclear magnetic resonance, high-performance liquid chromatography, and gel permeation chromatography.
Procurement of the heartelung blocks The surgical technique of procuring rabbit heartelungs for manipulation on an ex vivo circuit has been described, but a brief narrative is included. This surgical prep has been validated in several studies as an excellent model of ALI.18 A total of three 3-5 kg New Zealand white rabbits (Robinson Services, Inc, Mocksville, NC) are used for each experiment. One rabbit provides the heartelung block to be cannulated and ventilated on the ex vivo platform, whereas two others function as blood donors for the cardiopulmonary bypass circuit. After appropriate anesthesia is achieved with a combination of intramuscular ketamine (35 mg/kg) and xylazine (6.5 mg/kg) and intravenous acepromazine (5 mg/kg), a cut down tracheostomy is performed and median sternotomy incision is used to gain access to the thoracic cavity. The rabbit is ventilated on a volume control mode at a rate of 20 breaths per minute, a tidal volume of 10 mL/kg, and a fraction of inspired concentration of oxygen of 100% (Harvard ventilator apparatus, model 665; Harvard Apparatus, Holliston, MA). The superior vena cava, inferior vena cava, pulmonary artery, and aorta are identified and encircled with suture. Intracardiac prostaglandin (30 mg) and a bolus of intravenous heparin (1000 U/kg) are administered. A right ventriculotomy is fashioned, and an inflow cannula is advanced into the main pulmonary artery. An outflow cannula is then positioned in the left atrium and secured. The inferior vena cava, superior vena cava, and aorta are then ligated, instillation of 250 mL of cold (4 C) Perfadex (XVIVO, Goteborg, Sweden) via the inflow cannula is initiated and the heartelung block is dissected from the chest cavity (Fig. 1). All the animals in this experiment were treated in a humane fashion in accordance with our institutional animal care and use guidelines.
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Reperfusion of heartelung blocks After heparinization, two rabbits undergo exsanguination via a right ventricular cannula to provide 300 cc of blood for the bypass circuit. A left atrial cannula is introduced into the heartelung block and secured for blood pressure monitoring. The rabbit lung is ventilated with the aforementioned settings and the Sarns 5000 roller head pump cardiopulmonary bypass circuit (Sarns, Ann Arbor, MI) is de-aired.
Lung samples Lung tissue is removed at the following time points throughout the course of the experiment1: 1 h after the initiation of reperfusion and2 at the completion of the experiment (2-h reperfusion). Tissue is either frozen with liquid nitrogen and stored at 80 C for quantification or preserved in 4% paraformaldehyde for confocal imaging.
Determining the influence of dose, reperfusion duration, and injury status on D-FITC accumulation To determine the influence of the D-FITC reperfusion dose on lung uptake after reperfusion, two doses (30 or 150 mg) were bolused into the blood perfusate and allowed to continuously
circulate for the duration of the 2-h experiment. D-FITC tissue levels were then assessed at time point2 to determine the ultimate percent uptake. Based on this information, a D-FITC reperfusion dose was identified for subsequent parts of the experiment. To compare the uptake of dendrimer in injured and uninjured tissue after procurement, one set of heartelung blocks is cold stored (4 C) in Perfadex solution for 18 h (ischemic insult). A bolus of the D-FITC is then administered into the bypass circuit and allowed to continuously circulate for the duration of the 2-h experiment with tissue removed after 1- and 2-h reperfusion. Lungs randomized to the uninjured cohort underwent left atrial cannulation and ventilation and reperfusion immediately after the procurement without a cold-ischemic insult. The perfusate was bolused in a similar manner with D-FITC, and tissue was removed as outlined.
Quantification of D-FITC A novel fluorescence-based quantification method with high sensitivity (reaching 10 ng levels of detection) was used to measure dendrimer accumulation in the lung tissue removed at time points 1 and 2.19 Approximately 100 mg of lung tissue was homogenized in 1 mL of methanol in 2-mL DNA LoBind Eppendorf tubes (TissueLyser LT; Qiagen, Hilden, Germany). After homogenization, no intact cellular structures were observed under the microscope. Tissue homogenates were then diluted to 100 mg/mL and sonicated for 15 min to fully extract the D-FITC from the specimen. The resulting homogenates were centrifuged (15,000 rpm) at 4 C for 15 min to separate the supernatants from the tissue pellet. The fluorescence spectrum of the resulting supernatants was measured using a Shimadzu RF-5301 Spectrofluorophotometer (Shimadzu, Japan). D-FITC in each sample was quantified by comparing the measured fluorescence (lext ¼ 495 nm and lemi ¼ 520 nm), against an appropriate calibration curve. The ultimate tissue quantity was calculated by constructing a D-FITCebased calibration curve (R2 ¼ 0.98) of the recorded intensities (expressed as percentage of injected dose per gram of tissue [%ID/g]).
Immunofluorescence study of dendrimer distribution
Fig. 1 e Heartelung block on ex vivo circuit. The pulmonary artery cannula provides inflow (black-filled arrow) and the left atrial cannula outflow (white-filled arrow) on the cardiopulmonary bypass circuit. Tracheal intubation noted with white asterisk (*). (Color version of figure is available online.)
Procured tissues were fixed with 4% paraformaldehyde immediately after harvesting, sequentially processed with increasing concentrations of a sucrose solution (10%-30%) and cryosectioned. Tissues were sectioned transversely into 10mm thick slices using a Leica CM 1905 cryostat. These slices were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Life technology, Frederick, MD) and Tomatolectin-Texas Red (Vector Labs, Burlingame, CA). The samples were then imaged using a confocal LSM 710 microscope (Carl Zeiss, Hertfordshire, UK) under 20 and 40 magnification. For each image, settings were carefully optimized based on noneD-FITC perfused rabbit lung controls to avoid background fluorescence. Laser power, pinhole, gain, offset, and digital gain were selected separately for each magnification and kept consistent throughout the entire study.
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Fig. 2 e D-FITC accumulation in the ischemiaereperfusion lung represented in microgram (A) and as a percentage of the reperfusion dose (B). The weight of two rabbit lungs was 20 g. The low and high bolus doses were 30 and 150 mg, respectively. Two additional rabbits were needed for the 2-h, low bolus group to augment tissue quantity for the assay.
Results Dendrimer-FITC lung accumulation as a function of dose, time, and degree of injury To gain a better understanding of dendrimer uptake kinetics in this ex vivo model, we quantified D-FITC accumulation at different bolus doses, reperfusion durations, and injury status. Four experiments were performed to determine uptake after 1 h of reperfusion in both the low and the high bolus dose groups. An additional two experiments were performed in the
low bolus dose group to obtain enough sample for quantification. At the lower reperfusion dose (30 mg), 187-236 mg of D-FITC accumulated in the lung tissue, corresponding to 0.63%-0.79% of the reperfusion dose. When the initial reperfusion dose increased fivefold (150 mg), we found an increase of D-FITC accumulation to 343-586 mg (twofold increase). This demonstrates a decrease in the accumulation of D-FITC in relation to the perfused dose, indicating a possible ceiling for D-FITC uptake by the damaged lung (Fig. 2). Based on these findings, we identified a D-FITC reperfusion dose of 90 mg to investigate the effects of reperfusion time and injury status on D-FITC accumulation. Four experiments were performed in each arm of this portion of the study (both the control and experimental groups). Our findings demonstrate greater D-FITC uptake by injured lung tissue after 2 h of reperfusion. Specifically, after 1 h of reperfusion, both the injured and uninjured lungs exhibited similar D-FITC uptake (w0.20% of the perfused dose). After 2 h, however, D-FITC accumulation in injured lung specimen increased 100% (w0.40% of reperfusion dose), whereas remaining relatively unchanged in the uninjured lungs (w0.21%; Fig. 3).
Dendrimers target pneumocytes and alveoari macrophages
Fig. 3 e D-FITC accumulation in the control and injured lungs as a function of time. The weight of two rabbit lungs was 20 g. The bolus dose used in this experiment was 90 mg dendrimer per lung. The sample size for each group is 4.
As stated previously, one of the proposed therapeutic benefits of dendrimer-based delivery is its ability to selectively target the injured cells. Accordingly, we used immunofluorescence staining and confocal microscopy to determine the ultimate cellular distribution of the D-FITC. Interestingly, D-FITC was identified in the major histologic subtypes (epithelium and inflammatory) by the completion of reperfusion (Fig. 4). Based on anatomy and the cell morphology, we showed that, compared with the tissue background (Fig. 4A), the D-FITC colocalized with the pneumocytes in the alveoli (Fig. 4B) and bronchiole (Fig. 4C) from the injured lung tissue. In addition, we found some uptake of D-FITC by alveoli macrophages (Fig. 4D). Expectantly, the 1-h samples demonstrated a limited amount of D-FITC uptake at the cellular level. After 2 h of
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Fig. 4 e Confocal imaging of D-FITC distribution in the ischemiaereperfusion lung demonstrating targeted cell uptake. (A) Section without dendrimer reperfusion; (B) D-FITC accumulation in the pneumocytes; (C) D-FITC accumulation in the bronchiole; (D) D-FITC accumulation in the alveolar macrophages. The white arrow indicates dendrimer accumulation. The scale bar in the image is 20 mm. I: Alveoli; II: Pneumocytes; III: Bronchiole; IV: Alveoli macrophage. Red: Lectin, Green: D-FITC, Blue: DAPI. (Color version of figure is available online.)
reperfusion, however, D-FITC was identified in all cell types (Fig. 5).
Discussion The feasibility of intravenously introducing nanoparticles into an ex vivo model of ALI was previously unknown. We demonstrated that D-FITC is not only preferentially taken up by damaged lung tissue but that it also shows a timedependent accumulation in specific cellular targets. The latter could have profound implications on future dendrimerdrug conjugateebased therapies aimed at minimizing the morbidity and mortality of ALI. The pathogenesis of this inflammatory process is critically important when devising a successful delivery platform. The mechanism involves infiltration of the lung tissue by polymorphonuclear neutrophils whose local activation results in subsequent injury.7 As pro-inflammatory cytokines are
released, a cascade of events is initiated and ultimately leads to endothelial damage and increased capillary permeability.20 Previous studies on capillary permeability in ALI indicated variations in the degree of capillary damage based on the infiltration coefficient,21,22 which might hinder the extravasation of nanoparticles of a larger size at the site of injury. The small size (w4 nm in diameter) and nearly neutral surface charge (z-potential: þ4.5 0.1 mV) of the PAMAM dendrimer constructed for these experiments undoubtedly contributed to its ability to traverse the endothelium and gain access to its cellular targets.23 As prolonged reperfusion resulted in a greater severity of lung injury and further deterioration in cellto-cell adhesion, an expected increase in D-FITC accumulation was noted. This was not only true in the proximal airway and segmental bronchi, but also at the anatomic level of pulmonary gas exchange. Given the injury profile associated with ALI, dendrimerdrug conjugate nanotherapy could prove efficacious in affecting the major cellular components of repair while
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Fig. 5 e Confocal imaging demonstrating dendrimer accumulation in lung tissue before reperfusion and after 1 and 2 h of reperfusion. The area shown in the image was taken under 203 with tilescan and the sample size is approximately to 1 3 1 mm. The scale bar in the image is 100 mm. The white arrow indicates dendrimer accumulation. Red: Lectin, Green: DFITC, Blue: DAPI. (Color version of figure is available online.)
simultaneously minimizing systemic toxicity. Dendrimer nanoparticles have been previously shown to localize to damaged areas of the lung.16 More specifically, an investigation into direct pulmonary administration (via inhalation) of a PAMAM dendrimer-methylprednisolone conjugate for the treatment of asthma demonstrated that conjugated therapy resulted in a significant decrease in the concentration of inflammatory cells in comparison to free drug alone.15 There are several limitations, however, in extrapolating these findings, and assuming similar efficacy, in the treatment of ALI. First, and perhaps most importantly, the structural changes and inflammatory response associated with asthma are different in regards to their magnitude, duration, and cellular effectors than that experienced in ALI. In the latter, gross pulmonary edema most likely limits the biodistribution of inhaled dendrimer in the distal airway. Another study established the beneficial role of dendrimer therapy in mitigating the severe lung injuryeinflammation after LPS endotoxemia.16 Again, however, the gastrointestinal route of drug administration could limit the clinically applicability of this treatment in patients with extreme cases of ALI. Neither of the aforementioned studies explored the specific cellular localization of the dendrimer.
Although these prior investigations have demonstrated some success in ameliorating lung injury in animal models, not only can the route of delivery be clinically problematic but there is also a lack of understanding of the actual pathways involved in the anti-inflammatory effects. The present study represents the first known demonstration of the cellular-level localization of dendrimer in lung tissue following systemic administration. As stated, the dendrimer was identified in the major alveolar cell subtypes (epithelial and inflammatory). This extends the possibility of using dendrimer-based, conjugated therapies in multiple capacities in the inflammatory cascade of ALI. This should not be underemphasized as it is known that pneumocytes (epithelial cells) undergo shedding and alterations in function as a result of ALI.24 Previous studies have demonstrated that this pattern of injury in type II pneumocytes results in the production of dysfunctional surfactant, a phospholipid instrumental in maintaining normal alveolar surface tension.25,26 And, whereas previous attempts at exogenous surfactant administration in the treatment of ALI have yield inconsistent results, dendrimer conjugates could improve bioavailability and offer additional routes for drug administration.27-29 Similar strategies could be used to target circulating macrophages, which have not only been
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implicated in the development of ALI30,31 but also its resolution.32
references
Limitations
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In spite of the interesting cellular localization of the dendrimers, the findings of this experiment are based on a limited sample size. In addition, although IRI has been established as a reliable surrogate for ALI, we appreciate that subtle differences undoubtedly exist at the cellular level between these two processes. This could affect the degree of nanoparticle extravasation and targeted tissue uptake. Given that uptake in the control lungs was so trivial at the 1-h interval, and persisted at the 2-h interval, it was difficult to discern which cell types consistently took up the nanoparticle. Accordingly, it was not possible to describe differences in the cellular targets between these two arms of the experiment. Future studies are required to determine the systemic uptake profile of the dendrimer to minimize systemic toxicities in the other end organs that might sequester the nanoparticle.
Conclusion Although ALI confers substantial morbidity and mortality to a wide range of patient populations, current treatment options are limited. Nanoparticle-based therapy offers a promising new approach to target the known cellular effectors involved in both the inflammatory and reparative processes. To our knowledge, this is the first study to demonstrate the successful administration of intravenous dendrimer on an ex vivo IRI model of ALI, to identify targets of its uptake, and to quantify the influence of injury on tissue accumulation. Future studies into dendrimer-drug conjugates could have profound implications on the clinical management of this condition and warrant additional investigation.
Acknowledgment The authors would like to thank the Wilmer Core Module for Microscopy and Imaging for allowing us to use LSM710 confocal microscopy. The authors also want to acknowledge Melissa Jones and Jeffrey Braun for their attention to detail in all facets of this experiment, from intraoperative surgical technique and anesthesia to postoperative care. This work was funded by NIBIB R01EB018306 (R.K.). Authors’ contributions: J.C.G., T.C.C., J.T.M., and F.Z. did the data analysis; J.C.G., F.Z., K.N.R., and A.S.S. contributed in methodology; J.C.G. and F.Z. wrote the article; K.N.R. and A.S.S. approved the final version of the article.
Disclosures The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in the article.
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