An injectable thermoresponsive-hydrogel for lamellar keratoplasty: In-situ releases celastrol and hampers corneal scars.

Corneal stromal fibrosis is a common cause of visual impairment resulting from corneal injury, inflammation and surgery. Therefore, there is an unmet need for inhibiting corneal stromal fibrosis. However, bioavailability of topical eye drops is very low due to the tear and corneal barriers. In situ delivery offers a unique alternative to improve efficacy and minimize systemic toxicity. Herein, a drug delivery platform based on thermoresponsive injectable hydrogel/nano-micelles composite with in situ drug-controlled release and long-acting features is developed to prevent corneal scarring and reduce corneal stromal fibrosis in lamellar keratoplasty. The in-situ gelation hydrogels enabled direct delivery of celastrol to the corneal stroma. In vivo evaluation with a rabbit anterior lamellar keratoplasty model showed that hydrogel/micelles platform could effectively inhibit corneal stromal fibrosis. This strategy achieves controlled and prolonged release of celastrol in the corneal stroma of rabbit. Following a single corneal interlamellar injection, celastrol effectively alleviated fibrosis via mTORC1 signal promoting autophagy and inhibiting TGF-ß1/Smad2/3 signaling pathway. Overall, this strategy demonstrates promise for the clinical application of celastrol in preventing corneal scarring and reducing corneal stromal fibrosis post-lamellar keratoplasty, highlighting the potential benefits of targeted drug delivery systems in ocular therapeutics.

M2 macrophages promote subconjunctival fibrosis through YAP/TAZ signalling.

PURPOSE: To evaluate the role of M2 macrophages in subconjunctival fibrosis after silicone implantation (SI) and investigate the underlying mechanisms. MATERIALS AND METHODS: A model of subconjunctival fibrosis was established by SI surgery in rabbit eyes. M2 distribution and collagen deposition were evaluated by histopathology. The effects of M2 cells on the migration (using wound-scratch assay) and activation (by immunofluorescence and western blotting) of human Tenon's fibroblasts (HTFs) were investigated. RESULTS: There were more M2 macrophages (CD68+/CD206+ cells) occurring in tissue samples around silicone implant at 2 weeks postoperatively. Dense collagen deposition was observed at 8 weeks after SI. In vitro experiment showed M2 expressed high level of CD206 and transforming growth factor-ß1 (TGF-ß1). The M2-conditioned medium promoted HTFs migration and the synthesis of collagen I and fibronectin. Meanwhile, M2-conditioned medium increased the protein levels of TGF-ß1, TGF-ßR II, p-Smad2/3, yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ). Verteporfin, a YAP inhibitor, suppressedTGF-ß1/Smad2/3-YAP/TAZ pathway and attenuated M2-induced extracellular matrix deposition by HTFs. CONCLUSIONS: TGF-ß1/Smad2/3-YAP/TAZ signalling may be involved in M2-induced fibrotic activities in HTFs. M2 plays a key role in promoting subconjunctival fibrosis and can serve as an attractive target for anti-fibrotic therapeutics.

Carboxymethyl chitosan regulates macrophages polarization to inhibit early subconjunctival inflammation in conjunctival injury.

Persistent subconjunctival inflammation leads to subconjunctival fibrosis and eventual visual impairment. There is an unmet need for how to effectively inhibit subconjunctival inflammation. Herein, the effect of carboxymethyl chitosan (CMCS) on subconjunctival inflammation was investigated and the mechanism was involved. The evaluation of cytocompatibility demonstrated that CMCS had good biocompatibility. The in vitro results showed that CMCS inhibited secretions of pro-inflammatory cytokines (IL-6, TNF-α, IL-8 and IFN-γ) and chemokines (MCP-1), and downregulated TLR4/MyD88/NF-κB pathway in M1. The in vivo results displayed that CMCS alleviated conjunctival edema and congestion, and improved conjunctival epithelial reconstruction significantly. Both in vitro and in vivo results demonstrated that CMCS inhibited the infiltration of macrophages and reduced the expressions of iNOS, IL-6, IL-8 and TNF-α in the conjunctiva. Given that CMCS indicated the activities of inhibiting M1 polarization, NF-κB pathway, and subconjunctival inflammation, which may be employed as a potent treatment for subconjunctival inflammation.

Celastrol-based nanomedicine hydrogels eliminate posterior capsule opacification.

Aim: To formulate an injectable thermosensitive micelle-hydrogel hybrid system loaded with celastrol (celastrol-loaded micelle hydrogel: CMG) to prevent posterior capsule opacification (PCO). Materials & methods: Celastrol-loaded micelles were embedded in a thermosensitive hydrogel matrix to enable controlled on-demand celastrol delivery into the residual capsule. The efficacy and mechanisms of the system for eliminating PCO were evaluated in rabbits. Results: Celastrol-loaded micelles inhibited the migration and proliferation of lens epithelial cells induced by TGF-ß1. Celastrol prevents epithelial-mesenchymal transition in lens epithelial cells induced by TGF-ß1 through the TGF-ß1/Smad2/3/TEAD1 signaling pathway. In vivo efficiency evaluations showed that CMG demonstrated an excellent inhibitory effect on PCO in rabbits and had no obvious tissue toxicity. Conclusion: Injectable CMG may represent a promising ophthalmic platform for preventing PCO. This versatile injectable micelle-hydrogel hybrid represents a clinically relevant platform to achieve localized therapy and controlled release of drugs in other disease therapies.

Celastrol-based nanomedicine promotes corneal allograft survival.

Effectively promoting corneal allograft survival remains a challenge in corneal transplantation. The emerging therapeutic agents with high pharmacological activities and their appropriate administration routes provide attractive solutions. In the present study, a celastrol-loaded positive nanomedicine (CPNM) was developed to enhance corneal penetration and to promote corneal allograft survival. The in vitro, in vivo and ex vivo results demonstrated the good performance of CPNM prolonging the retention time on ocular surface and opening the tight junction in cornea, which resulted in enhanced corneal permeability of celastrol. Both in vitro and in vivo results demonstrated that celastrol inhibited the recruitment of M1 macrophage and the expression of TLR4 in corneal allografts through the TLR4/MyD88/NF-κB pathway, thereby significantly decreasing secretion of multiple pro-inflammatory cytokines to promote corneal allograft survival. This is the first celastrol-based topical instillation against corneal allograft rejection to provide treatment more potent than conventional eye drops for ocular anterior segment diseases.

Co-Delivery of Paclitaxel and siRNA with pH-Responsive Polymeric Micelles for Synergistic Cancer Therapy.

Due to the complex physiological characteristics of tumors, chemotherapy or gene therapy alone cannot completely kill tumor cells. Therefore, combining chemotherapy with gene therapy for combination therapy is the key to solving this problem. However, there are still significant challenges in how to simultaneously deliver and rapidly release the drugs and siRNA into cancer cells. In this work, a triblock copolymer was synthesized to co-deliver siRNA and paclitaxel to tumor cells. This system has an acid-sensitive subsurface layer, which can not only load siRNA to prevent premature drug release but also has good controlled release performance. In vitro experiments showed that polymeric vectors can efficiently deliver siRNA and paclitaxel simultaneously into tumor cells for rapid release within the tumor cells. This study reveals that this novel polymeric micelle is a suitable vector for the codelivery of chemotherapeutic drugs and siRNA to cancer cells, representing an important advance in nanotechnology, nanomedicine, drug delivery, and cancer therapy.

Hypoxia-Responsive Polymeric Micelles for Enhancing Cancer Treatment.

Polymeric drug vectors have shown great potentials in cancer therapy. However, intelligent controlled release of drugs has become a major challenge in nanomedicine research. Hypoxia-responsive polymeric micelles have received widespread attention in recent years due to the inherent hypoxic state of tumor tissue. In this study, a novel diblock polymer consisting of polyethylene glycol and poly[glutamic acid (3-(2-nitro-imidazolyl)-propyl)] was synthesized and self-assembled into hypoxia-responsive polymeric micelles for the controlled release of doxorubicin (DOX). The cell experiments demonstrated that DOX-loaded micelles had a stronger killing capacity on tumor cells under hypoxic conditions, while the blank micelles had good biocompatibility. All the experiments indicate that our hypoxia-responsive polymeric micelles have a great potential for enhanced cancer treatment.

Effectively suppressed angiogenesis-mediated retinoblastoma growth using celastrol nanomicelles.

Celastrol, a Chinese herbal medicine, has already shown an inhibition effect on retinoblastoma growth activity in our previous research, but its mechanism is not well understood. Angiogenesis is a main driving force in many tumors. Here, we studied whether celastrol could inhibit angiogenesis-mediated retinoblastoma growth, if so, through what mechanism. In this work, we developed celastrol-loaded polymeric nanomicelles to improve the poor water solubility of celastrol. When given an intraperitoneal injection to mice bearing human retinoblastoma xenografts, celastrol nanomicelles (CNMs, 27.2 mg/kg/2 days) significantly reduced the weight and the volume of tumors and decreased tumor angiogenesis. We found that CNMs suppressed hypoxia-induced proliferation, migration, and invasion by human umbilical vascular endothelial cells (EA.hy 926) in a dose-dependent manner. Furthermore, CNMs inhibited SO-Rb 50 cells-induced sprouting of the vessels and vascular formation in chick embryo chorioallantoic membrane assay in vitro. To understand the molecular mechanism of these activities, we assessed the signaling pathways in CoCl2 treated EA.hy 926. CNMs inhibited the hypoxia-induced HIF-1α and VEGF. In conclusion, our results reveal that CNMs target the HIF-1α/VEGF pathway, which may be an important reason for the suppression of retinoblastoma growth and angiogenesis.

Antiangiogenic and Antitumor Therapy for Retinoblastoma with Hypoxia-Inducible Factor-1α siRNA and Celastrol Co-Delivery Nanomicelles.

Retinoblastoma (RB) makes up about 3% of all childhood malignancies. Chemotherapy is commonly applied for RB treatment, while the clinical effectiveness varies significantly due to the cancer therapeutic resistances. Hypoxic tumor microenvironment, a hallmark of all tumors, is strongly associated with malignant progress and therapeutic resistances. The hypoxia mainly promotes the angiogenesis by upregulating pro-angiogenetic pathways. In this work, polymeric micelles are used as the carrier to deliver celastrol and siRNA to RB cells for achieving synergistic anti-tumor and antiangiogenesis effects. The micelle vectors have shown effective cellular internalization and release of loaded-celastrol and HIF-1 siRNA. The co-delivery system specifically and synergistically inhibits the expression of HIF-1α and VEGF in RB cells, suppresses the HIF-1α /VEGF/VEGFR signaling pathway, and impedes the proliferation, migration, and invasion of vascular endothelial cells. The polymer micellar carrier that co-delivers HIF-1α siRNA and celastrol is used for antiangiogenic and antitumor therapy of RB. Altogether, the results show that our polymeric micelle delivery system can be used to overcome barriers of drug resistance induced by angiogenesis and develop new drug/siRNA combinatory therapies.

PiggyBac transposon system with polymeric gene carrier transfected into human T cells.

CAR-T cell-based immunotherapy has shown great promise in clinical trials for the treatment of hematological malignancies. The majority of these trials utilize retroviral and lentiviral vectors to introduce CAR transgene. In spite of its satisfactory efficiency, the concerns about the potential carcinogenicity and complicated synthesis procedure restrict widespread clinical applications of viral vectors. Recent studies show that transposon-based gene transfer is a safer and simpler non-viral approach for stable transgene expression. Here, we developed an in house made polymeric nanomicelles carrier for piggyBac (PB) transposon delivery to primary T lymphocytes. The properties, transfection efficiency and toxicity of this carrier was analyzed. Results indicated that nanomicelles produced in our study were stable and reduction-sensitive. These micelles can completely condense DNA and mediate transfection with efficiency of average 30.2% with high cell viability (> 80%). Furthermore, incorporating piggyBac transposase elements into polyplexes promoted persistent expression of the transgene (up to 55%). At the end of culture, CAR-T cells mainly exhibited memory phenotype and consisted of CD3+CD8+ T cells. The cytotoxicity of these CAR-T cells was average 17% at 20:1 ratio. In conclusion, polymeric nanomicelles provide a flexible and safe method for gene delivery to T lymphocytes.

Multifunctional Polymeric Carrier for Co-Delivery of MRI Contrast Agents and siRNA to Tumors.

Gene therapy holds tremendous promise in the treatment of tumors, in which an effective delivery system plays a crucial role. Herein, a nanoscale polymeric micelle is developed to co-deliver superparamagnetic iron oxide nanoparticles (SPIONs) and siRNA targeting luciferase gene (siLuc) into tumors. In vitro studies showed that SPIO and siRNA-loaded micelles could enter cancer cells easily and they have no cytotoxicity. And in vivo studies indicated that polymeric micelle could deliver SPIO and siRNA into tumors efficiently. Our results strongly demonstrated that the novel polymeric micelle is a promising carrier for delivery of siRNA and SPIO to enable noninvasive imaging-assisted therapy in vivo.

Fabrication and Biocompatibility of Core-Shell Structured Magnetic Fibrous Scaffold.

In recent years, various magnetic bio-materials with physical and morphological cues have been used in biomedical area due to their advantageous characteristics. As one class of them, magnetic fibrous scaffolds have attracted many researchers' interests because they have an important positive impact on cellular growth behavior. They provide various physical cues to regulate the regeneration and repair of damaged tissue, and promote new tissue formation. In this study, we developed core-shell structured magnetic fibers (3D CS-MFs) using cooperative assembly method the combination with electrospinning technology. The obtained magnetic 3D CS-MFs displayed excellent magnetic performance, biocompatibility, and provided a desirable microenvironment for model cells (bone marrow mesenchymal stem cells, BMSCs) growth. More importantly, BMSCs exhibited excellent viability and 3D growth. The novel preparation method will greatly enhance the potential application of magnetic fibrous scaffold in the biomedical area, such as drug release, and tissue engineering.

Synergistic Effects of Electrical Stimulation and Aligned Nanofibrous Microenvironment on Growth Behavior of Mesenchymal Stem Cells.

Incontrollable cellular growth behavior is a significant issue, which severely affects the functional tissue formation and cellular protein expression. Development of natural extracellular matrix (ECM) like biomaterials to present microenvironment cues for regulation of cell responses can effectively overcome this problem. The external simulation and topological characteristics as typical guiding cues are capable of providing diverse influences on cellular growth. Herein, we fabricated two-dimensional aligned conductive nanofibers (2D-ACNFs) by an electrospinning process and surface polymerization, and the obtained 2D-ACNFs provided the effects of both alignment and electrical stimulation (ES) on cellular response of human mesenchymal cells (hMSCs). The results of cellular responses implied that the obtained 2D-ACNFs could offer a synergistic effect of both ES and aligned nanopattern on hMSC growth behavior. The effects could not only promote hMSCs to contact each other and maintain cellular activity but also provide positive promotion to regulate cellular proliferation. Thus, we believe that the obtained 2D-ACNFs will have a broad application in the biomedical field, such as cell culture with ES, directional induction for cell growth, and damaged tissue repair, etc.

A DOX-loaded polymer micelle for effectively inhibiting cancer cells.

A novel triblock polymer is synthesized and self-assembled with doxorubicin to form DOX-loaded micelles. The synthetic process involves the ring-opening polymerization, carboxylation and amidation reactions, and the structures are characterized. The drug release test indicated that the micelles have the ability to control the release of drugs. The cell uptake results indicated that the DOX-loaded micelles could enter cancer cells easily, and the cytotoxicity and apoptosis test confirmed that DOX-loaded micelles have a strong killing effect on tumor cells, while the blank micelles do not have cytotoxicity. Therefore, the novel polymer micelles are a promising carrier for delivery of anticancer drugs to enhance cancer treatment.

Celastrol nanomicelles attenuate cytokine secretion in macrophages and inhibit macrophage-induced corneal neovascularization in rats.

The aim of the present study was to investigate the inhibitory effects of celastrol-loaded nanomicelles (CNMs) on activated macrophage-induced corneal neovascularization (CNV) in rats and cytokine secretion in macrophages. Using an angiogenesis assay in vitro, we detected the effects of CNMs on human umbilical vein endothelial cell (HUVEC) migration and invasion. In addition, the expression levels of cytokines secreted from hypoxia-induced macrophages were assessed through cytokine array analysis. The expression of hypoxia-inducible factors-1α (HIF-1α), nuclear factor-kappa B p65 (NF-κB p65), phospho-nuclear factor-kappa B p65 (phospho-NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-ERK1/2 was analyzed by western blotting. Activated macrophages were elicited through mineral oil lumbar injection, labeled with 1,19-dioctadecyl-3-3-39,39-tetramethylindocarbocyanine (DiI) and implanted into the corneal micro-pocket to induce CNV and to assess the antiangiogenic effect in rats. CNV was morphometrically analyzed using ImageJ software. Histopathological features were evaluated by immunofluorescence immunostaining for vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) on day 2 after surgery. In the present study, the results indicated that CNMs significantly inhibited the migration and invasion of HUVECs; remarkably attenuated the expression of VEGF, tumor necrosis factor-α, interleukin-1α, monocyte chemoattractant protein 1, cytokine-induced neutrophil chemoattractant 3, and MMP-9 protein; and downregulated ERK1/2, p38 MAPK, NF-κB activation, and HIF-1α expression in macrophages. The peritoneal cells elicited using mineral oil were highly purified macrophages, and the length and area of CNV were significantly decreased in the CNMs group compared with the control group. There was a significant reduction in the expression of VEGF and MMP-9 in activated macrophages and corneal tissue after pretreatment with CNMs in this model. In conclusion, CNMs potently suppressed macrophage-induced CNV via the inhibition of VEGF and MMP-9 expression. This effect might be mediated through attenuating macrophages via HIF-1α, MAPK, and NF-κB signaling pathways.

Synthesis and Characterization of pH-Responsive Copolypeptides Vesicles for siRNA and Chemotherapeutic Drug Co-Delivery.

Novel diblock copolypeptides are synthesized and evaluated as a pH-sensitive drug delivery vector for anticancer drugs and siRNA co-delivery. The synthetic process involve the ring opening polymerization of α-amino acid N-carboxyanhydrides as well as aminolysis and deprotection reactions, and the structures are characterized. The copolypeptides can self-assemble into stable vesicles at neutral pH, and disassemble in acidic endosomal/lysosomal, which shown the pH-responsive behavior. The cell uptake results indicated that the vesicles can co-deliver DOX and siRNA inside the same cell and exhibit a pH-sensitive release behavior. Therefore, the novel polymeric vesicles are a promising carrier for co-delivery anticancer drug and siRNA to overcome the drug resistance and enhance cancer treatment.

Doxycycline-mediated inhibition of corneal angiogenesis: an MMP-independent mechanism.

PURPOSE: We explored the underlying mechanisms of doxycycline inhibition of corneal angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro in the presence or absence of VEGF, doxycycline, or broad-spectrum matrix metalloproteinase (MMP) inhibitors (1,10-phenanthroline [1PT] and batimastat). HUVEC proliferation was determined using Cell Counting Kit-8. Rats with VEGF-induced corneal neovascularization (CNV) were treated with saline solution, 0.1% doxycycline, 0.1% 1PT, or 50 µM batimastat (n = 7/group). The length and area of CNV were measured on day 7. The activity of MMP-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blotting and ELISA were used to analyze the expression of phosphorylated eNOS and Akt, and PI3K activity. RESULTS: Our results showed that doxycycline inhibited HUVEC proliferation induced by VEGF in a dose-dependent manner in vitro, and decreased CNV induced by VEGF in vivo in terms of vessel length and area. 1PT and batimastat showed similar MMP inhibitory functions with doxycycline in vitro and in vivo, but they had no effects on HUVEC proliferation, and only partially mimicked the inhibitory properties of doxycycline (∼45%) on angiogenesis induced by VEGF. In addition, although doxycycline is capable of modulating the PI3K/Akt-eNOS pathway in vitro and in vivo in an MMP-independent manner, 1PT and batimastat were not. CONCLUSIONS: The mechanism of doxycycline-mediated inhibition of angiogenesis occurs through MMP inhibitory activity and modulation of the PI3K/Akt-eNOS pathway, an MMP-independent mechanism.

Activated macrophages induce neovascularization through upregulation of MMP-9 and VEGF in rat corneas.

PURPOSE: To explore the mechanisms of activated macrophages (A-Mφ) involved in corneal angiogenesis. METHODS: Activated macrophages were elicited by mineral oil lumbar injection and implanted into corneal micropockets in rats for the treatment group, A-Mφ, and phosphate-buffered saline group as control. Corneal changes were observed with a slit lamp microscope, and histopathological features were evaluated by immunofluorescence. Reverse transcription-polymerase chain reaction was used to detect the relative expression of angiogenesis-associated factors and inflammatory mediators in the activated macrophages and corneal tissue after implantation. RESULTS: Immunofluorescence showed that peritoneal cells expressed antigens of cluster of differentiation 68 (CD68, ED1), matrix metalloproteinases-9 (MMP-9), and vascular endothelial growth factor (VEGF). Activated macrophages significantly induced corneal neovascularization (CNV), which peaked on day 5, whereas the control group and normal corneas showed less CNV. The activated macrophages and corneal tissue after implantation expressed the angiogenesis-related factors, such as cyclooxygenase-2, platelet-derived growth factor, transforming growth factor beta, interleukin-1 alpha, MMP-9, and VEGF in messenger RNA (mRNA). However, mRNA expression of MMP-9 and VEGF differed significantly only in the cornea between the A-Mφ group and phosphate-buffered saline group 5 days after the implantation. MMP-9 and VEGF expression of mRNA and protein was higher in the A-Mφ group than that in the control group and normal corneas. CONCLUSIONS: Activated macrophages induce obvious CNV and related mechanisms, which may be correlated with MMP-9 and VEGF autocrine in activated macrophages and upregulation of MMP-9 and VEGF in corneal tissue.

Antitumor activity of celastrol nanoparticles in a xenograft retinoblastoma tumor model.

BACKGROUND: Celastrol, a Chinese herbal medicine, has shown antitumor activity against various tumor cell lines. However, the effect of celastrol on retinoblastoma has not yet been analyzed. Additionally, the poor water solubility of celastrol restricts further therapeutic applications. The goal of this study was to evaluate the effect of celastrol nanoparticles (CNPs) on retinoblastoma and to investigate the potential mechanisms involved. METHODS: Celastrol-loaded poly(ethylene glycol)-block-poly(ɛ-caprolactone) nanopolymeric micelles were developed to improve the hydrophilicity of celastrol. The 2-(2-methoxy-4- nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulf-ophenyl)-2H tetrazolium monosodium salt (WST-8) assay was used to determine the inhibitory effect of CNPs on SO-Rb 50 cell proliferation in vitro. Immunofluorescence was used to evaluate the apoptotic effect of CNPs on nuclear morphology, and flow cytometry was used to quantify cellular apoptosis. The expression of Bcl-2, Bax, NF-κB p65, and phospo-NF-κB p65 proteins was assessed by Western blotting. A human retinoblastoma xenograft model was used to evaluate the inhibitory effects of CNPs on retinoblastoma in NOD-SCID mice. Hematoxylin and eosin staining was used to assess the apoptotic effects of CNPs on retinoblastoma. RESULTS: CNPs inhibit the proliferation of SO-Rb 50 cells in a dose- and time-dependent manner with an IC(50) of 17.733 µg/mL (celastrol-loading content: 7.36%) after exposure to CNPs for 48 hours. CNPs induce apoptosis in SO-Rb 50 cells in a dose-dependent manner. The expression of Bcl-2, NF-κB p65, and phospo-NF-κB p65 proteins decreased after exposure to CNPs 54.4 µg/mL for 48 hours. Additionally, the Bax/Bcl-2 ratio increased, whereas the expression of Bax itself was not significantly altered. CNPs inhibit the growth of retinoblastoma and induce apoptosis in retinoblastoma cells in mice. CONCLUSION: CNPs inhibit the growth of retinoblastoma in mouse xenograft model by inducing apoptosis in SO-Rb 50 cells, which may be related to the increased Bax/Bcl-2 ratio and the inhibition of NF-κB. CNPs may represent a potential alternative treatment for retinoblastoma.

Celastrol nanoparticles inhibit corneal neovascularization induced by suturing in rats.

PURPOSE: Celastrol, a traditional Chinese medicine, is widely used in anti-inflammation and anti-angiogenesis research. However, the poor water solubility of celastrol restricts its further application. This paper aims to study the effect of celastrol nanoparticles (CNPs) on corneal neovascularization (CNV) and determine the possible mechanism. METHODS: To improve the hydrophilicity of celastrol, celastrol-loaded poly(ethylene glycol)-block-poly(ɛ-caprolactone) nanopolymeric micelles were developed. The characterization of CNPs was measured by dynamic light scattering and transmission electron microscopy analysis. Celastrol loading content and release were assessed by ultraviolet-visible analysis and high performance liquid chromatography, respectively. In vitro, human umbilical vein endothelial cell proliferation and capillary-like tube formation were assayed. In vivo, suture-induced CNV was chosen to evaluate the effect of CNPs on CNV in rats. Immunohistochemistry for CD68 assessed the macrophage infiltration of the cornea on day 6 after surgery. Real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were used to evaluate the messenger ribonucleic acid and protein levels, respectively, of vascular endothelial growth factor, matrix metalloproteinase 9, and monocyte chemoattractant protein 1 in the cornea. RESULTS: The mean diameter of CNPs with spherical shape was 48 nm. The celastrol loading content was 7.36%. The release behavior of CNPs in buffered solution (pH 7.4) showed a typical two-phase release profile. CNPs inhibited the proliferation of human umbilical vein endothelial cells in a dose-independent manner and suppressed the capillary structure formation. After treatment with CNPs, the length and area of CNV reduced from 1.16 ± 0.18 mm to 0.49 ± 0.12 mm and from 7.71 ± 0.94 mm(2) to 2.29 ± 0.61 mm(2), respectively. Macrophage infiltration decreased significantly in the CNP-treated corneas. CNPs reduced the expression of vascular endothelial growth factor, matrix metalloproteinase 9, and monocyte chemoattractant protein 1 in the cornea on day 6 after suturing. CONCLUSION: CNPs significantly inhibited suture-induced CNV by suppressing macrophage infiltration and the expression of vascular endothelial growth factor and matrix metalloproteinase 9 in the rat cornea.