Inflammation-Targeted Delivery of Celastrol via Neutrophil Membrane

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Inflammation-targeted Delivery of Celastrol via Neutrophil Membrane Coated Nanoparticles in the Management of Acute Pancreatitis Xu Zhou, Xi Cao, He Tu, Zhi-Rong Zhang, and Li Deng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01342 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 13, 2019

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Molecular Pharmaceutics

Figure 1. Size-dependent accumulation of PEG-PLGA NPs in the pancreas of AP rats. (A) Size distributions of NP60, NP150, NP300. (B) Biodistributions of NPs in AP rat model. Ex vivo fluorescence imaging of vital organs was performed at 1 h and 3 h after the i.v. injection of NP60, NP150, NP300. (C) Semi-quantitative analysis of fluorescence intensity of the pancreas. Data represent mean ± S.D. (n = 3). * p < 0.05 vs. DiD, # p < 0.05 vs. NP60, & p < 0.05 vs. NP150.

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Figure 2. NNPs/DiD selectively accumulated in inflamed pancreas. Ex vivo imaging was performed at 1 h and 3 h after i.v. injection of DiD solution, NPs/DiD and NNPs/DiD in AP rats. Ex vivo imaging of vital organs (A) and the pancreas (B). (C) Semi-quantitative analysis of fluorescence intensity of the pancreas. Data represent mean ± S.D. (n = 3). * p < 0.05 vs. DiD, # p < 0.05 vs. NPs/DiD. (D) Laser scanning confocal microscopy images of pancreatic tissue cryosections. Red represents DiD, and blue represents cell nuclei. Scale bar = 100 μm.


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Figure 3. Laser scanning confocal microscopy images of pancreas tissue cryosections obtained from AP rats. DiD, red; CD34, green, representing vessels; cell nuclei, blue (DAPI). Scale bar = 100 μm.



Figure 4. Therapeutic efficacy on local and systemic inflammation in the AP rat model. Serum amylase (A), ascites (B), and pancreatic wet/dry ratio (C) in AP rats at 3 h after i.v injections of CLT solution, NPs/CLT and NNPs/CLT at an equivalent dose of 1 mg/kg CLT. Data represent mean ± S.D. (n = 6). *p < 0.05 vs. sham, #p < 0.05 vs. model, &p < 0.05 vs. CLT, and $p < 0.05 vs. NPs/CLT. 243x60mm (300 x 300 DPI)


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Figure 5. Therapeutic efficacy on local and systemic inflammation in the AP rat model. Serum TNF-α (A) and serum IL-6 (B) levels in the AP rat model at 3 h after i.v injections of CLT solution, NPs/CLT and NNPs/CLT at an equivalent dose of 1 mg/kg CLT. Data represent mean ± S.D. (n = 6). *p < 0.05 vs. sham, #p < 0.05 vs. model, &p < 0.05 vs. CLT, and $p < 0.05 vs. NPs/CLT. 167x59mm (300 x 300 DPI)



Figure 6. Therapeutic efficacy on local and systemic inflammation in the AP rat model. Pancreatic MPO activity (A), lung MPO activity (B) in the AP rat model at 3 h after i.v injections of CLT solution, NPs/CLT and NNPs/CLT at an equivalent dose of 1 mg/kg CLT. Data represent mean ± S.D. (n = 6). *p < 0.05 vs. sham, #p < 0.05 vs. model, &p < 0.05 vs. CLT, and $p < 0.05 vs. NPs/CLT. 172x59mm (300 x 300 DPI)


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Figure 7. Immunohistochemistry of IL-6, NIK, IL-1β, NF-κB, IKK, CD126, TAK1 and TNF-α staining of pancreatic tissue sections in AP rats. Scale bar, 100 μm.



Figure 8. In vivo toxicity and therapeutic effect in AP rats. (A) Major organs and tissues were collected at 3 h post injection of indicated formulations, and subject to H&E staining and histological analysis. These are representative sections from five rats analyzed for each condition. Scale bar, 200 μm. (B) Serum ALT and AST levels in the AP rat model. Data represent mean ± S.D. (n = 5). *p < 0.05 vs. sham, #p < 0.05 vs. saline.


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graphic abstract



Submission to Molecular Pharmaceutics

Inflammation-targeted Delivery of Celastrol via Neutrophil Membrane Coated Nanoparticles in the Management of Acute Pancreatitis

Xu Zhou†, ‡, Xi Cao‡, He Tu†, Zhi-Rong Zhang‡, Li Deng*,‡

†

‡

Sichuan Provincial Orthopedic Hospital, Chengdu, 610041, China

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China

*Correspondence: Li Deng (L. Deng) Key Laboratory of Drug Targeting and Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University No 17, Section 3, Southern Renmin Rd, Chengdu, 610041, China Email: [email protected] Tel/Fax: +86-28-85503198


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ABSTRACT: Celastrol (CLT)-loaded PEG-PLGA nanoparticles (NPs/CLT) coated with neutrophil membranes (NNPs/CLT) were explored for the management of acute pancreatitis. PEG-PLGA nanoparticles sized around 150 nm were proven to selectively accumulate in the pancreas in rats with acute pancreatitis (AP). NNPs were found to overcome the blood-pancreas barrier and specifically distributed to the pancreatic tissues. Moreover, NNPs showed more selective accumulation in the pancreas than nanoparticles without any membrane coating in AP rats. Compared to CLT solution and NPs/CLT group, NNPs/CLT significantly downregulated the levels of pancreatic amylase and pancreatic myeloperoxidase in AP rats. Also, using NNPs as the delivery vehicle significantly reduced the systemic toxicity of CLT in AP rats. Together, these results suggest that NNPs/CLT represent a highly promising delivery vehicle for the targeted therapy of acute pancreatitis.

KEYWORDS: Acute pancreatitis; Celastrol; Neutrophil membrane; PEG-PLGA nanoparticles



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INTRODUCTION Acute pancreatitis (AP) is a sudden disease with severe abdominal pain, which often results

in inflammation of regional tissues and induces systemic inflammatory responses to generate severe life-threatening conditions.1,

2

When it comes to severe acute pancreatitis (SAP),

inflammatory mediators are released from overreacted leukocytes to cause cascade reactions and systemic inflammation leading to multiple organ dysfunction syndromes, especially the acute lung injury.3-6 Despite decades of intensive studies, no effective yet safe therapeutic options are available in clinic.1 This is likely due to the presence of blood-pancreas barrier (BPB) that significantly limits the anti-inflammatory drugs from accumulating in the pancreas site thus resulting in a poor therapeutic effect.7-9 Therefore, finding an alternative treatment option is critical to the effective management of AP. Despite the attempts that have been made for the treatment of AP, current AP interventions mainly include fluid resuscitation, analgesia, and early enteral feeding. As for drug therapy, antibiotics are often used for the prevention of pancreatic infections.10 With the understanding of pancreatitis, the suppression of inflammatory reaction has been the key to treat pancreatitis.6 Nuclear factor kappa B (NF-κB) transcription factors play a critical role in coordinating immune responses and inflammation.11, 12 Thus, anti-inflammatory drugs which specifically inhibit NF-κB activation are recommended for the managment of pancreatitis. Celastrol (CLT) is a chemical compound isolated from the root of Thunder God Vine, which has been well demonstrated to inhibit NF-κB activation and exhibit various anti-inflammatory, antioxidant and anticancer activities.13-16 Thus, CLT was chosen as a model compound for the treatment of AP in our study.


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Previous studies focused on the prodrug strategy to overcome BPB, and very few studies are available on using nanoparticles for achieving targeted therapy of AP.8, 17, 18 For instance, gadolinium nanoparticles based MRI probe was fabricated only for the diagnosis of early AP.18 Our group previously reported using N,N,N′-trimethyl-benzyl-1,3-propanediamine-Rhein to achieve targeted therapy of AP.8 Also, CLT conjugated with N, N-dimethyl-1,2-diaminoethane was synthesized and proven to achieve pancreas targeted delivery against SAP.17 Despite the enhanced uptake and distribution in the inflamed pancreas tissues, small molecule candidates require complex chemical synthesis and purification steps. Similar to the enhanced permeability and retention (EPR) effect at the tumor site, nanoparticulate delivery systems with certain sizes are proven to selectively accumulate at the inflammation site via the extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration (ELVIS) effect.1921

Wang et al once fabricated PCL-PEG micelles to deliver dexamethasone for the targeted

therapy of rheumatoid arthritis based on the ELVIS effect.22 Besides, HPMA nanoparticles conjugated with fluorescent dyes were successfully applied in the detection of inflammatory osteolysis, of which ELVIS-mediated mechanism was involved.23 Thus, delivering therapeutic cargos selectively to the pancreas via nanoparticles may serve as a neat and efficient delivery strategy. Nanoscale delivery systems coated with plasma membranes offer a unique delivery strategy for the targeted therapy of cancers and related diseases.24-27 For instance, cancer cell membrane biomimetic nanoparticles have been shown to selectively target tumor cells to achieve precision cancer imaging and therapy in vivo.28 However, no reports on using plasma membrane coated nanoparticles are available for the treatment of acute pancreatitis.



As the most abundant leukocytes in the circulation, neutrophils are rapidly recruited to the site of inflammation and provide the first line of defense against infections.29-31 Stimulated by cytokines, neutrophil granulocytes arriving at the inflammatory site are abnormally activated, disintegrate rapidly, and die in the way of “neutrophil extracellular traps (NETs)”.29, 32 Herein, we hypothesize that drug-loaded nanoparticles coated with neutrophil membranes can be driven to the inflammatory site via chemokine recruitment to achieve targeted anti-inflammatory therapy. In our study, CLT has been loaded in PEG-PLGA nanoparticles (NPs/CLT), and further coated with neutrophil membranes (NNPs/CLT). Using AP rats, biodistribution and pharmacodynamic studies were performed to evaluate the inflammation-specific accumulation in pancreas tissues and the therapeutic effects of NNPs/CLT in alleviating AP-related injuries in vivo.

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MATERIALS AND METHODS Materials. Poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide)

(PEG-PLGA50:50, PEG average Mw 5000 Da; PLGA 50:50, Mw 45000 – 75000 Da) was obtained from University of Electronic Science and Technology of China (Chengdu, China). CLT was provided by Chengdu Must Biotechnology (Chengdu, China). Amylase assay kit and myeloperoxidase (MPO) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other chemicals used were of analytical grade and purchased commercially.


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Animals. Male Sprague-Dawley rats weighing between 180 and 220 g were provided by Sichuan Provincial People's Hospital (Chengdu, China). All animal studies were performed per the university guidelines, and approved by the Ethics Committee of Sichuan University. Preparation and characterization of PEG-PLGA Nanoparticles (NPs) with three different particle sizes. NPs with three size distributions were fabricated by emulsion and solvent evaporation method.33 Specifically, nanoparticles with average particle sizes of 60 nm (NP60), 150 nm (NP150), and 300 nm (NP300) were prepared by changing the ultrasonication intensity and time. Biodistribution of NPs with different particle sizes in acute pancreatitis (AP) rats. AP rat models were established as described previously.17 In brief, male Sprague-Dawley rats were given 1% sodium pentobarbital (45 mg/kg, i.p.), which were then subjected to sterile laparotomy. The rats were then infused 3% sodium taurocholate in the pancreatic duct to induce acute pancreatitis.8 AP rats were randomly divided into four groups with six rats in each group: DiD solution, NP60/DiD, NP150/DiD and NP300/DiD. Nanoparticle suspensions and DiD solutions were administered intravenously (the concentration of DiD was 75 mg/kg) immediately after the induction of AP. At predetermined time points of 1 h and 3 h, rats were sacrificed, the vital organs and pancreas were excised for ex vivo fluorescence imaging by IVIS Spectrum imaging system (Perkin Elmer, USA). Preparation of Neutrophil Membrane Coated NPs (NNPs). NNPs were fabricated by coating NPs with neutrophil membranes via a direct extrusion method.34, 35



Biodistribution in AP Rats. Male Sprague-Dawley AP rats were randomly divided into four groups (n = 6): sham operated group, DiD group, NPs/DiD group, and NNPs/DiD group. DiD, NPs/DiD and NNPs/DiD were administered intravenously to the rats (the concentration

of DiD was 75 mg/kg) after the induction of AP. Rats in the sham operated group were infused with 0.9% normal saline as control. Rats were sacrificed at predetermined time points. The selected time points were based on the disease kinetics.8 After 1 h and 3 h, rats were sacrificed, samples of vital organs and pancreas were excised for ex vivo fluorescence imaging by IVIS Spectrum imaging system (PerkinElmer, USA). Pancreas tissues collected at 3 h were then fixed with 4% paraformaldehyde in PBS. Consecutive frozen sections (10 μm) were prepared for the evaluation whether the formulations could target the neovasculature. The vasculature was marked by CD34 and DAPI was used for cell nuclei staining. The frozen sections of pancreas were observed under a laser scanning confocal microscope (LSM710, Carl Zeiss, Germany). Pharmacodynamics. AP rats were randomly divided into five groups (n = 6): sham operated, model, CLT solution, NPs/CLT, and NNPs/CLT. Three CLT-containing formulations were administered intravenously to the rats at the equivalent dose of 1 mg/kg CLT) after the induction of AP. Rats in the sham operated group and model group were infused with 0.9% normal saline. Amylase activity in plasma was measured by the amylase assay kit per manufacturer’s instruction. The absorbance at the wavelength of 660 nm was then measured using Varioskan Flash microplate reader (Thermo Scientific, USA). The concentrations of pro-inflammatory cytokines including tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were determined using enzyme-linked immunosorbent assay


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(ELISA) kit per manufacturer’s instruction. Serum samples were added to each well which was already coated with antibody, and maintained at 37 °C for 30 min. Horseradish peroxidase (HRP)-conjugate reagent were added to incubate individual wells for another 0.5 h at 37 °C before the addition of tetramethylbenzidine (TMB) solution. In the end, the reaction was stopped by adding phosphoric acid and the absorbance was determined at the wavelength of 450 nm. The activity of myeloperoxidase (MPO) was determined using MPO assay kit per manufacturer’s instruction to evaluate neutrophil infiltration.8 Immunohistochemistry and Histological Analysis. To furtherly evaluate the therapeutic efficacy and the safety of all groups, AP rats were sacrificed at 3 h post injection of indicated formulations, then vital organs and pancreas of AP rats were collected. All samples were fixed with 10% neutral buffered formalin and embedded in paraffin, which were then subjected to hematoxylin and eosin staining (H&E) to evaluate the pathological changes. To evaluate the levels of inflammatory factors, immunohistochemical staining was performed on paraffin embedded pancreas sections. IL-6, TNF-α, TNK1, NIK, IL-1β and IL6R/CD126 as pro-inflammatory cytokines, IKK and NF-κB as downstream inflammatory factors were stained accordingly.34 All tissue sections were examined under light microscope (Zeiss Axiovert 40CFL, Germany). Statistical analysis. One-way ANOVA analysis was performed for comparing multiple groups followed by a Tukey post hoc analysis (Origin, USA). All data were presented as mean ± standard deviation (SD). In all cases, p < 0.05 was considered statistically significant.



RESULTS AND DISCUSSION



PEG-PLGA nanoparticles (NPs) and their size-dependent targeting in AP rats. To select an appropriate nanoparticle with appropriate size for the therapy of acute pancreatitis,36, 37 firstly PEG-PLGA nanoparticles of different size distributions ranging from 60 nm to about 300 nm were prepared (Figure 1A): NP60, an average particle size distribution of 61.4 ± 2.8 nm with a polydispersity index (PDI) of 0.175 ± 0.019; NP150, an average particle size distribution of 156.8 ± 2.3 nm with a PDI of 0.146 ± 0.018; NP300, an average particle size distribution of 303.7 ± 1.3 nm (PDI = 0.169 ± 0.047). All three formulations showed similar size distributions and a negative zeta potential of about -24.5 mV. Next, the distribution profiles of PEG-PLGA NPs in AP rat models were evaluated. The ex vivo fluorescence imaging of vital organs was performed at 1 h, and 3 h after systemic administration of NP60/DiD, NP150/DiD, NP300/DiD and DiD solution at an equivalent DiD dose. DiD solution distributed mainly in the liver and the lung, whereas DiD-loaded NPs of all sizes showed higher accumulation in the pancreas in AP rats. Interestingly, NP150 displayed enhanced pancreas accumulation with higher fluorescence intensity than NPs60 and NPs300 (Figure 1B and Figure 1C), which was extremely likely due to the ELVIS-mediated accumulation. Although ELVIS effect is mainly explored in the treatment of arthritis in recent studies, ELVIS effect may also exist in acute pancreatitis due to the presence of abundant inflammatory cells and the vascular leakage in the pancreatic tissues.19, 22 Thus, NP150 was then selected for the following study and the characterization of NNPs was studied in our previous study.34


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Figure 1. Size-dependent accumulation of PEG-PLGA NPs in the pancreas of AP rats. (A) Size distributions of NP60, NP150, NP300. (B) Biodistributions of NPs in AP rat model. Ex vivo fluorescence imaging of major organs was performed at 1 h and 3 h after the i.v. injection of NP60, NP150, NP300. (C) Semi-quantitative analysis of fluorescent intensity within the pancreas. * p < 0.05 versus DiD solution group, # p < 0.05 versus NP60/DiD group,

&

p < 0.05 versus

NP150/DiD group. Data represent mean ± S.D. (n = 3).

Biodistribution of NNPs in AP Rats. To explore biodistribution profile of NNPs, acute pancreatitis models were established in Sprague-Dawley rats. Next, ex vivo fluorescence imaging of major organs was performed at 1 h and 3 h after intravenous injection of three DiDcontaining formulations. The biodistribution of the nanoparticles in major organs and pancreas at the given time points was shown in Figure 2A. DiD solution distributed throughout major organs, and mainly accumulated in liver, spleen and lung. In comparison, the accumulations of NNPs/DiD and NPs/DiD in the spleen were decreased and fluorescent intensity in the spleen



and lung was distinctly increased (Figure 2A). Moreover, NNPs/DiD showed significantly increased accumulation in the pancreas compared to both DiD solution and NPs/DiD group (Figure 2B and 2C). As expected, NNPs/DiD showed the highest distributions in the inflamed pancreatic tissues among all groups. Moreover, the accumulation of NNPs/DiD appeared to increase over time. This phenomenon further indicated that nanoparticles coated with neutrophil membranes could overcome the blood-pancreas barrier and be selectively taken up by inflammatory pancreatic tissues thus representing a promising strategy to achieve targeted delivery to pancreas. Interestingly, NNPs/DiD also showed up-regulated accumulations in the lung, which could be highly desirable because lung injury is a common symptom in patients with acute pancreatitis.3, 38 Acute pancreatitis may lead to systemic inflammatory responses especially with lung, thus making lung tissues suitable targets for treatment of acute pancreatitis.39 Therapeutic strategies which can simultaneously deliver drugs to both pancreas and lung tissues have recently been reported as promising approaches in the management of acute pancreatitis.8, 17 Thus, the biodistribution profile of NNPs supported that NNPs might be a promising strategy to achieve dual-pancreas and lung targeting. The therapeutic mechanism of NNPs treating acute pancretitis is based on the fact NNPs have been shown to be selectively internalized by inflammatory cells,34 which may result in enhanced cellular uptake efficiency at the site of inflammation. To explore whether NNPs could target inflamed pancreas by targeting the neovasculature, we measured the overlap of NNPs and the vasculature in the pancreas. CD34, a biomarker of vasculature, showed extensive distribution across the inflamed pancreas tissue (Figure 3). Besides, NNPs/DiD showed extensive distributions across the pancreatic tissue, not only accumulating around the vessel


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areas, but also overcoming the vessel wall and penetrating into the parenchymal region of the pancreas. Overall, the result demonstrated that the therapeutic mechanism did not involve in neovasculature-targeting, and that nanoparticles coated with neutrophil membranes could most likely be recruited by chemokines and driven to the inflammatory site.

Figure 2. NNPs/DiD selectively accumulated in inflamed pancreas. (A) Ex vivo fluorescent imaging of major organs in AP rats at 1 h and 3 h. (B) Ex vivo fluorescent imaging of pancreas in AP rats at 1 h and 3 h. (C) Semi-quantitative analysis of fluorescent intensity within the pancreas. * p < 0.05 versus DiD solution group,

#

p < 0.05 versus NPs/DiD group. Data



represent mean ± S.D. (n = 3). (D) Laser scanning confocal microscopy images of pancreatic tissue cryosections. Red represents DiD, and blue represents cell nuclei. Scale bar = 100 μm.

Figure 3. Laser scanning confocal microscopy images of pancreas tissue cryosections obtained from AP rats. DiD, red; CD34, green, representing vessels; cell nuclei, blue (DAPI). Scale bar = 100 μm. Therapeutic efficacy in the AP rat model. Next, the therapeutic efficacy of CLT formulations was evaluated in AP rats. Amylase activity is considered as the key indicator of acute pancreatitis.8 As expected, NNPs/CLT treatment group significantly inhibited the activity of pancreatic amylase (Figure 4A), compared to CLT solution and NPs/CLT group (p < 0.05).

Ascites, as a clinical symptom in the development of multiple organ dysfunction syndrome, was quantified to evaluate the severity of AP.40 As shown in Figure 4B, after the treatment of NNPs/CLT, the weight of ascitic fluids in AP rats were obviously lower than model group (p < 0.05), while CLT solution and NPs/CLT didn’t ameliorate the symptom (p  0.05). Additionally, we examined pancreas wet/dry ratios from AP rats to


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determine the pancreatic edema.17 Compared with the other three groups (model group, CLT solution treatment group and NPs/CLT treatment group), NNPs/CLT treatment group showed significantly decreased the weight of ascites and the wet/dry ratio (Figure 4B and 4C) (p < 0.05), indicating decreased severity of pancreatic edema, while no obvious differences were detected in CLT solution, NPs/CLT and model group. Histological analysis of pancreas after the treatment of NNPs showed decreased inflammatory cell infiltration and acinar cell necrosis, as compared to CLT solution treated group and NPs/CLT treated group (Figure 8A), indicating the reduced tissue damage of pancreas. Taken together, all results suggest that NNPs/CLT successfully alleviated local pancreatic inflammation, attributing to the overcoming of the blood-pancreas barrier and selectively accumulating in the inflammatory pancreas.

Figure 4. Therapeutic efficacy of NNPs/CLT on inflammation in the AP rat model. Serum amylase level (A), weight of ascites (B), and pancreatic wet/dry ratio (C) in AP rats. *p < 0.05 versus sham group, #p < 0.05 versus model group, &p < 0.05 versus CLT solution group, and $p < 0.05 versus NPs/CLT group. Data represent mean ± S.D. (n = 6).



Figure 5. Therapeutic efficacy of NNPs/CLT on inflammation in the AP rat model. Serum TNF-α (A) and serum IL-6 (B) levels in the AP rat model. *p < 0.05 versus sham group, #p < 0.05 versus model group, &p < 0.05 versus CLT solution group, and $p < 0.05 versus NPs/CLT group.

Figure 6. Therapeutic efficacy of NNPs/CLT on inflammation in the AP rat model. Pancreatic MPO activity (A), lung MPO activity (B) in the AP rat model. *p < 0.05 versus sham group, #p < 0.05 versus model group, &p < 0.05 versus CLT solution group, and $p < 0.05 versus NPs/CLT group. Systemic inflammation were mainly caused by the inflammatory cytokines released from injured pancreatic acinar cells.41 To evaluate the systemic anti-inflammatory therapeutic effect of NNPs/CLT, serum levels of TNF-α and IL-6 were determined.42 Inflammatory cytokines such as TNF-α and IL-6 are important indicators of the disease progression of AP, thus making


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it necessary to elucidate the changes of TNF-α and IL-6 levels during the treatment.2,

39

Compared to model group, CLT solution failed to efficiently degrade TNF-α levels in serum (p  0.05), while NPs/CLT and NNPs/CLT successfully reduced TNF-α levels in serum (p < 0.05) (Figure 5). NNPs/CLT showed the best effect among the three treatment groups. Similarly, CLT solution and NPs/CLT couldn’t efficiently reduce serum levels of IL-6 (p  0.05), whereas NNPs/CLT significantly decreased them, whether compared to model group or CLT solution and NPs/CLT group (p < 0.05). All the results demonstrated an immediate anti-inflammatory effect of NNPs/CLT against inflammation with systemic injury. Besides, as shown in Figure 6, lung MPO activity and pancreas MPO activity were dramatically decreased after treatment of NNPs/CLT (p < 0.05), while myeloperoxidase (MPO) activity indicated neutrophil infiltration in the pancreas and lungs.17 Moreover, H&E staining showed NNPs/CLT markedly reduced pulmonary alveolar thickening and inflammatory cell infiltration in the lungs which were induced by acute pancreatitis (Figure 8A), which further indicated NNPs/CLT help control the inflammation in the lung. Immunohistochemistry analysis of inflammatory cytokines. Per immunohistological analysis, the local level of IL-6 in the pancreas was significantly downregulated after NNPs/CLT treatment (Figure 7), which was consistent with the plasma results (Figure 5), whereas the level of TNF-α in the pancreas did not show significant differences across treatment groups (Figure 7). Overall, these results suggest that NNPs/CLT could inhibit inflammation by down-regulating both the serum levels of pro-inflammatory cytokines TNF-α and IL-6 and possibly the local level of IL-6 thus alleviating systemic and local inflammation.



Besides the level of TNF-α and IL-6,we also performed the immunohistological analysis of pro-inflammatory cytokines such as IL-1β, NF-κB, CD126 and TAK1.43, 44 As shown in Figure 7, immunohistochemistry revealed that the NNPs/CLT treated group displayed less proinflammatory cytokines in the pancreas area as compared to CLT, NPs/CLT and saline treated groups. The expression of pro-inflammatory cytokines such as TNF-α and IL-6 is proven to be regulated by the nuclear factor NF-κB, thereby NNPs/CLT could possibly inhibit the expression of NF-κB to further block the signaling pathway to upregulate the pro-inflammatory cytokines.45 The decrease of inflammatory factors in the pancreas indicated that the symptoms of acute pancreatitis had been relieved and the therapeutic effect on pancreatitis worked.


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Figure 7. Immunohistochemistry images of pancreatic tissues in AP rats. Scale bar, 100 μm.

In vivo toxicity in AP rat model. Although CLT shows strong anti-inflammatory activities, CLT is also known to exhibit potent toxicity such as reproductive toxicity.46, 47 As a result, it is necessary to explore whether NNPs/CLT could reduce the adverse side effects in AP rats. As shown in Figure 8, besides the therapeutic effect on the pancreas and lung as mentioned before,



CLT solution showed severe cardiac toxicity and hepatotoxicity, which was observed by myocardial fiber breakage and necrocytosis.

Serum ALT levels of CLT solution group were

significantly higher than sham group (p < 0.05), furtherly demonstrating hepatotoxicity of CLT solution. On the contrary, both NPs/CLT and NNPs/CLT group did not show obvious sign of cardiac toxicity and hepatotoxicity, indicating nanoparticulate drug delivery systems may help reduce the systemic toxicity of CLT. This phenomenon was likely due to the enhanced accumulation of NNPs/CLT in the pancreas, which lead to a reduced distribution in other organs.


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Figure 8. In vivo toxicity and therapeutic effect in AP rats. (A) Histological analysis of H&E staining tissues which were collected at 3 h post injection of indicated formulations. Scale bar, 200 μm. (B) Serum ALT and AST levels in the AP rat model. *p < 0.05 versus sham group, #p < 0.05 versus saline group. Data represent mean ± S.D. (n = 5).



CONCLUSION



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In summary, nanoparticles coated with neutrophil membranes have been shown to selectively accumulate in the pancreas in AP rats. Specifically, NNPs/CLT have been well demonstrated to markedly reduce the activity of pancreatic amylase and reduce associated lung injuries in the acute pancreatitis rat model. Moreover, NNPs/CLT could reduce relevant proinflammatory cytokines both locally and systemically and reduce systemic toxicity of CLT. Taken together, CLT delivered via neutrophil membrane coated nanoparticles represents a highly promising strategy against acute pancreatitis.



ACKNOWLEDGEMENTS Authors are very grateful for the financial support from the Natural Science Foundation of

China (81690261).

 1.

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For Table of Contents Use Only

Manuscript title: Inflammation-targeted Delivery of Celastrol via Neutrophil Membrane Coated Nanoparticles in the Management of Acute Pancreatitis Authors: Xu Zhou, Xi Cao, He Tu, Zhi-Rong Zhang, Li Deng*


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Inflammation-Targeted Delivery of Celastrol via Neutrophil Membrane

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