Abstract
Dysregulated mucosal immune responses and colonic fibrosis impose two formidable challenges for ulcerative colitis treatment. It indicates that monotherapy could not sufficiently deal with this complicated disease and combination therapy may provide a potential solution. A chitosan-modified poly(lactic-co-glycolic acid) nanoparticle (CS-PLGA NP) system was developed for co-delivering patchouli alcohol and simvastatin to the inflamed colonic epithelium to alleviate the symptoms of ulcerative colitis via remodeling immune microenvironment and anti-fibrosis, a so-called “two-birds-one-stone” nanotherapeutic strategy. The bioadhesive nanomedicine enhanced the intestinal epithelial cell uptake efficiency and improved the drug stability in the gastrointestinal tract. The nanomedicine effectively regulated the Akt/MAPK/NF-κB pathway and reshaped the immune microenvironment through repolarizing M2Φ, promoting regulatory T cells and G-MDSC, suppressing neutrophil and inflammatory monocyte infiltration, as well as inhibiting dendritic cell maturation. Additionally, the nanomedicine alleviated colonic fibrosis. Our work elucidates that the colon-targeted codelivery for combination therapy is promising for ulcerative colitis treatment and to address the unmet medical need.
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Introduction
Ulcerative colitis (UC) is the major form of inflammatory bowel disease (IBD). UC is a relapsing and remitting mucosal inflammation that starts from the rectum and spreads continuously to the proximal segments of the colon [1]. The prevalence of UC has been rising in the newly industrialized countries in Africa, Asia, and South America [2]. For example, it is about 11.6 per 100,000 people in China [3]. Additionally, the incidence of colorectal cancer in Asian patients with UC has been also increasing [4]. This situation imposes a great need for effective UC treatment, but the current therapy methods cannot meet the expectations due to the unsustainable efficacy [5].
The maladjustment of the immune system affected by heredity, environment, and gut microbiota is closely related to the progress of UC [6]. The intestinal immune microenvironment consists of intestinal epithelial cells, macrophages, dendritic cells (DCs), regulatory T cells (Tregs), and inflammatory T cells, which collaboratively maintain immune homeostasis [7]. The inflammatory microenvironment can be a target for UC treatment. Macrophages (MΦ) are pivotal in coordinating the progress of UC [8]. Macrophages are characterized by their diversity and plasticity in response to environmental signals and are traditionally classified into M1Φ with pro-inflammatory/anti-microbial activity and M2Φ with anti-inflammatory activity/tissue repair [9]. An increase of M1Φ amount in the pathological site of colitis predicts the worsening disease stage [10]. Re-education from M1 to M2 phenotype is a potential strategy for UC treatment [11].
In addition to the aggravated inflammatory immune responses in colitis, excessive proliferation of fibroblasts and myofibroblasts contributes to the deposition of extracellular matrix (ECM) and the fibrosis of the intestinal wall. Severe intestinal fibrosis may result in intestinal obstruction and require surgical intervention [12]. Traditional treatments mainly focus on alleviating the symptoms of UC through anti-inflammatory approaches (e.g., 5-aminosalicylic acid, corticosteroids, immunosuppressants, or monoclonal antibodies), but their clinical application has been restrained because of unsustainable therapeutic effect, the recurrence after drug withdrawal, and off-target systemic side effects [1]. Moreover, these medications are of little help in solving the intestinal fibrosis problem that is a complication of UC [12]. Therefore, the synergy of immune regulation and anti-fibrosis may be a new strategy for UC treatment.
To address this issue, we proposed a combination therapy strategy using an oral nanomedicine for co-delivering patchouli alcohol (PA) and simvastatin (SV), a “two-birds-one-stone” nanotherapeutic strategy. We previously revealed that patchouli alcohol, a natural tricyclic sesquiterpene isolated from a Chinese herb Guang Huo ** a safe and effective drug for UC. The interaction between the inflammatory immune microenvironment and colitis-related fibrosis during the progression of UC has not been fully demonstrated yet, and further investigation and understanding will be helpful to better depict the underlying mechanisms and seek effective drug combinations.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional file.
Abbreviations
- MTT:
-
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide
- CS:
-
Chitosan
- JNK:
-
C-Jun NH2 terminal kinase
- COX-2:
-
Cyclooxygenase-2
- DCs:
-
Dendritic cells
- DSS:
-
Dextran sodium sulfate
- DiR:
-
1,1-Dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide
- DL:
-
Drug-loading capacity
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- EE:
-
Encapsulation efficiency
- ECM:
-
Extracellular matrix
- ERK1/2:
-
Extracellular signal-regulated kinase
- FBS:
-
Fetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- GC–FID:
-
Gas chromatography–flame ionization detector
- G-MDSC:
-
Granulocytic myeloid-derived suppressor cells
- H&E:
-
Hematoxylin/eosin
- HPLC:
-
High-performance liquid chromatography
- iNOS:
-
Inducible nitric oxide synthase
- IBD:
-
Inflammatory bowel disease
- IPM:
-
Inflammatory peritoneal macrophages
- IFN-γ:
-
Interferon γ
- IL-4:
-
Interleukin-4
- LPS:
-
Lipopolysaccharide
- M-CSF:
-
Macrophage colony-stimulating factor
- MAPK:
-
Mitogen-activated protein kinases
- NF-κB:
-
Nuclear factor-κB
- PA:
-
Patchouli alcohol
- PI3K:
-
Phosphatidylinositol-3 kinases
- PLGA:
-
Poly (lactic-co-glycolic acid)
- PDI:
-
Polydispersity index
- PVA:
-
Polyvinyl alcohol
- Akt:
-
Protein kinase B
- ROS:
-
Reactive oxygen species
- qPCR:
-
Real-time quantitative polymerase chain reaction
- Tregs:
-
Regulatory T cells
- SV:
-
Simvastatin
- CMC-Na:
-
Sodium carboxymethyl cellulose
- TGF-β:
-
Transforming growth factor-β
- TEM:
-
Transmission electron microscope
- TNF-α:
-
Tumor necrosis factor α
- UC:
-
Ulcerative colitis
- XRD:
-
X-ray diffractometer
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Acknowledgements
We thank the Molecular Imaging Center and TEM Facility at SIMM and the National Center for Protein Science Shanghai, CAS for the technical support.
Funding
National Key Research and Development Program of China (2021YFE0103100, 2021YFC2400600); NFSC (81925035, 8201101172, and 81803736); Shanghai SciTech Innovation Initiative (19431903100, 18430740800).
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JZ: investigation, methodology, data curation, formal analysis, visualization, writing—original draft. AO: investigation, validation, formal analysis, data curation. XT, RW, YZ, PZ: investigation, resources. YF: investigation, validation. YF, DC: investigation, data curation. BW: project administration, supervision. YH: conceptualization, project administration, formal analysis, writing—review and editing. All authors read and approved the final manuscript.
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All the animal experimental procedures were complied with the institutional ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC), Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC No. SYXK2015-0027).
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Supplementary Information
Additional file 1: Figure S1.
Effect of PA on macrophage repolarization and synergistic effect with SV. (A–C) The mRNA levels of M1-associated pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) in PA-treated RAW264.7 macrophages, as measured by qPCR. (D, E) Western blot analysis of Akt/MAPK/NF-κB pathway-related biomarkers and M2-related MR expression after PA treatment. (F) The mRNA levels of the M1-related pro-inflammatory molecules (e.g., TNF-α, iNOS, and COX-2) and M2-related Arg1 in SV-treated RAW264.7 macrophages, as measured by qPCR. (G) IL-6 mRNA levels in LPS-induced peritoneal macrophages treated with PA (10 μM) and SV (0, 1, and 2 μM). Figure S2. Cytotoxicity study of PA and SV on (A) Caco-2 cells, (B) RAW264.7 cells, and (C) L929 cells. Cytotoxicity of the NPs in (D) L929 cells and (E) M2Φ. Figure S3. Anti-colitis treatment of synergistic drugs. (A) Schematic diagram of DSS-induced colitis and treatment. (B) Changes in daily bodyweight of each group during the trial period. (C) Statistical analysis and (D) images of colon lengths in each group (n = 4). Figure S4. Preliminary biosafety assessment of PA and SV. (A) Organ coefficients. (B) H&E staining of the major organs (Scale bar: 100 μm). Figure S5. Fluorescence images of (A) M1Φ and (B) L929 after incubation with the coumarin 6-labeled NPs (scale bar: 50 µm). (C, E) Histogram and (D, F) mean fluorescence intensity of the NPs-internalized M1Φ (LPS-induced RAW264.7 cells) and L929 cells were analyzed by flow cytometry (n = 3). Figure S6. Specific accumulation of CS-PLGA NPs in inflamed colons. Ex vivo imaging and radiant efficiency of (A, B) organs and (C) colons at 3 h. Ex vivo imaging and radiant efficiency of (D, E) organs and (F) colons at 5 h (n = 3). Figure S7. (A–E) Individual bodyweight curves and (F–J) DAI curves in CMC-Na, DSS, PA/SV, PLGA NPs, or CS-PLGA NPs groups (n = 6). Figure S8. Preliminary biosafety assessment. (A) Organ coefficients. (B) H&E staining of the major organs (Scale bar: 100 μm). Figure S9. The dot plots of M2Φ and DCs in the colon tissue. Figure S10. The dot plots of neutrophils, inflammatory monocytes, and G-MDSCs in the colon tissue. Figure S11. The dot plots of Tregs in the colon tissue. Table S1. The primer sequence used in qPCR. Table S2. Disease activity index (DAI) scoring. Table S3. Characterization of the NPs. Table S4. Drug encapsulation efficiency and drug loading efficacy.
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Zhang, J., Ou, A., Tang, X. et al. “Two-birds-one-stone” colon-targeted nanomedicine treats ulcerative colitis via remodeling immune microenvironment and anti-fibrosis. J Nanobiotechnol 20, 389 (2022). https://doi.org/10.1186/s12951-022-01598-0
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DOI: https://doi.org/10.1186/s12951-022-01598-0