Enhanced radiotherapy using photothermal therapy based on dual-sensitizer of gold nanoparticles with acid-induced aggregation.

The damaged DNA strands caused by radiotherapy (RT) can repair by themselves. A gold nanoparticles (GNPs) system with acid-induced aggregation was developed into a dual sensitizer owing to its high radioactive rays attenuation ability and enhanced photothermal heating efficiency after GNPs aggregation to achieve a combination therapy of RT and photothermal therapy (PTT). In this combination therapy, the formed GNP aggregates firstly showed a higher sensitize enhancement ratio (SER) value (1.52). Importantly, the self-repair of damaged DNA strands was inhibited by mild PTT through down-regulating the expression of DNA repair protein, thus resulting in a much higher SER value (1.68). Anti-tumor studies further demonstrated that this combination therapy exhibited ideal anti-tumor efficacy. Furthermore, the imaging signals of GNPs in computed tomography and photoacoustic were significantly improved following the GNPs aggregation. Therefore, a dual sensitizer with multimodal imaging was successfully developed and can be further applied as a new anti-tumor therapy.

A Carrier-Free Nanostructure Based on Platinum(IV) Prodrug Enhances Cellular Uptake and Cytotoxicity.

Flurbiprofen, a hydrophobic COX inhibitor, was coordinated axially with oxoplatin to form a new conjugate, cis, cis, trans-[Pt(IV)(NH3)2Cl2(flurbiprofen)2]. The successful synthesis of this new conjugate was confirmed by 1H, 13C, and 195Pt NMR. The potential of this conjugate being reduced to cisplatin and subsequently exerting its DNA cross-linking ability was verified using cyclic voltammetry (CV), HPLC, and mass spectrometry (MS). This conjugate showed markedly higher cytotoxicity on many cancer cell lines than cisplatin, flurbiprofen, and their physical mixture (mole ratio, cisplatin:flurbiprofen = 1:2). This is consistent with the result of an apoptosis-inducing assay. This conjugate spontaneously assembles carrier-free nanoparticles in aqueous solution, which is confirmed by DLS, TEM, SEM, and AFM, and thus facilitates cellular uptake and markedly improves its cytotoxicity and apoptosis-inducing ability in vitro.

Ultrasmall Gold Nanoparticles Behavior in Vivo Modulated by Surface Polyethylene Glycol (PEG) Grafting.

Ultrasmall nanoparticles provide us with essential alternatives for designing more efficient nanocarriers for drug delivery. However, the fast clearance of ultrasmall nanoparticles limits their application to some extent. One of the most frequently used compound to slow the clearance of nanocarriers and nanodrugs is PEG, which is also approved by FDA. Nonetheless, few reports explored the effect of the PEGylation of ultrasmall nanoparticles on their behavior in vivo. Herein, we investigated the impact of different PEG grafting level of 2 nm core sized gold nanoparticles on their biological behavior in tumor-bearing mice. The results indicate that partial (∼50%) surface PEGylation could prolong the blood circulation and increase the tumor accumulation of ultrasmall nanoparticles to a maximum extent, which guide us to build more profitable small-sized nanocarriers for drug delivery.

Poly(vinyl methyl ether/maleic anhydride)-Doped PEG-PLA Nanoparticles for Oral Paclitaxel Delivery To Improve Bioadhesive Efficiency.

Bioadhesive nanoparticles based on poly(vinyl methyl ether/maleic anhydride) (PVMMA) and poly(ethylene glycol) methyl ether-b-poly(d,l-lactic acid) (mPEG-b-PLA) were produced by the emulsification solvent evaporation method. Paclitaxel was utilized as the model drug, with an encapsulation efficiency of up to 90.2 ± 4.0%. The nanoparticles were uniform and spherical in shape and exhibited a sustained drug release compared with Taxol. m-NPs also exhibited favorable bioadhesive efficiency at the same time. Coumarin 6 or DiR-loaded nanoparticles with/without PVMMA (C6-m-NPs/DiR-m-NPs or C6-p-NPs/DiR-p-NPs) were used for cellular uptake and intestinal adhesion experiments, respectively. C6-m-NPs were shown to enhance cellular uptake, and caveolae/lipid raft mediated endocytosis was the primary route for the uptake of the nanoparticles. Favorable bioadhesive efficiency led to prolonged retention in the intestine reflected by the fluorescence in isolated intestines ex vivo. In a ligated intestinal loops model, C6-m-NPs showed a clear advantage for transporting NPs across the mucus layer over C6-p-NPs and free C6. The apparent permeability coefficient (Papp) of PTX-m-NPs through Caco-2/HT29 monolayers was 1.3- and 1.6-fold higher than PTX-p-NPs and Taxol, respectively, which was consistent with the AUC0-t of different PTX formulations after oral administration in rats. PTX-m-NPs also exhibited a more effective anticancer efficacy, with an IC50 of 0.2 ± 1.4 µg/mL for A549 cell lines, further demonstrating the advantage of bioadhesive nanoparticles. The bioadhesive nanoparticles m-NPs demonstrated both mucus permeation and epithelial absorption, and thus, this bioadhesive drug delivery system has the potential to improve the bioavailability of drugs that are insoluble in the gastrointestinal environment.

Targeting tumor microenvironment with PEG-based amphiphilic nanoparticles to overcome chemoresistance.

Multidrug resistance is one of the biggest obstacles in the treatment of cancer. Recent research studies highlight that tumor microenvironment plays a predominant role in tumor cell proliferation, metastasis, and drug resistance. Hence, targeting the tumor microenvironment provides a novel strategy for the evolution of cancer nanomedicine. The blooming knowledge about the tumor microenvironment merging with the design of PEG-based amphiphilic nanoparticles can provide an effective and promising platform to address the multidrug resistant tumor cells. This review describes the characteristic features of tumor microenvironment and their targeting mechanisms with the aid of PEG-based amphiphilic nanoparticles for the development of newer drug delivery systems to overcome multidrug resistance in cancer cells. FROM THE CLINICAL EDITOR: Cancer is a leading cause of death worldwide. Many cancers develop multidrug resistance towards chemotherapeutic agents with time and strategies are urgently needed to combat against this. In this review article, the authors discuss the current capabilities of using nanomedicine to target the tumor microenvironments, which would provide new insight to the development of novel delivery systems for the future.

Cerebrospinal fluid efflux through dynamic paracellular pores on venules as a missing piece of the brain drainage system.

The glymphatic system plays a key role in the clearance of waste from the parenchyma, and its dysfunction has been associated with the pathogenesis of Alzheimer's disease (AD). However, questions remain regarding its complete mechanisms. Here, we report that efflux of cerebrospinal fluid (CSF)/interstitial fluid (ISF) solutes occurs through a triphasic process that cannot be explained by the current model, but rather hints at the possibility of other, previously undiscovered routes from paravenous spaces to the blood. Using real-time, in vivo observation of efflux, a novel drainage pathway was discovered, in which CSF molecules enter the bloodstream directly through dynamically assembled, trumpet-shaped pores (basolateral ϕ<8 µm; apical ϕ < 2 µm) on the walls of brain venules. As Zn2+ could facilitate the brain clearance of macromolecular ISF solutes, Zn2+-induced reconstruction of the tight junctions (TJs) in vascular endothelial cells may participate in pore formation. Thus, an updated model for glymphatic clearance of brain metabolites and potential regulation is postulated. In addition, deficient clearance of Aß through these asymmetric venule pores was observed in AD model mice, supporting the notion that impaired brain drainage function contributes to Aß accumulation and pathogenic dilation of the perivascular space in AD.

Liposome-based multifunctional nanoplatform as effective therapeutics for the treatment of retinoblastoma.

Photothermal therapy has the characteristics of minimal invasiveness, controllability, high efficiency, and strong specificity, which can effectively make up for the toxic side effects and tumor resistance caused by traditional drug treatment. However, due to the limited tissue penetration of infrared light, it is difficult to promote and apply in clinical practice. The eye is the only transparent tissue in human, and infrared light can easily penetrate the eye tissue, so it is expected that photothermal therapy can be used to treat fundus diseases. Here in, a new nano-platform assembled by liposome and indocyanine green (ICG) was used to treat retinoblastoma. ICG was assembled in liposomes to overcome some problems of ICG itself. For example, ICG is easily quenched, self-aggregating and instability. Moreover, liposomes can prevent free ICG from being cleared through the systemic circulation. The construction of the nano-platform not only ensured the stability of ICG in vivo, but also realized imaging-guide photothermal therapy, which created a new strategy for the treatment of retinoblastoma.

An amphiphilic dendrimer as a light-activable immunological adjuvant for in situ cancer vaccination.

Immunological adjuvants are essential for successful cancer vaccination. However, traditional adjuvants have some limitations, such as lack of controllability and induction of systemic toxicity, which restrict their broad application. Here, we present a light-activable immunological adjuvant (LIA), which is composed of a hypoxia-responsive amphiphilic dendrimer nanoparticle loaded with chlorin e6. Under irradiation with near-infrared light, the LIA not only induces tumour cell lysis and tumour antigen release, but also promotes the structural transformation of 2-nitroimidazole containing dendrimer to 2-aminoimidazole containing dendrimer which can activate dendritic cells via the Toll-like receptor 7-mediated signaling pathway. The LIA efficiently inhibits both primary and abscopal tumour growth and induces strong antigen-specific immune memory effect to prevent tumour metastasis and recurrence in vivo. Furthermore, LIA localizes the immunological adjuvant effect at the tumour site. We demonstrate this light-activable immunological adjuvant offers a safe and potent platform for in situ cancer vaccination.

Comprehensive understanding of magnetic hyperthermia for improving antitumor therapeutic efficacy.

Magnetic hyperthermia (MH) has been introduced clinically as an alternative approach for the focal treatment of tumors. MH utilizes the heat generated by the magnetic nanoparticles (MNPs) when subjected to an alternating magnetic field (AMF). It has become an important topic in the nanomedical field due to their multitudes of advantages towards effective antitumor therapy such as high biosafety, deep tissue penetration, and targeted selective tumor killing. However, in order for MH to progress and to realize its paramount potential as an alternative choice for cancer treatment, tremendous challenges have to be overcome. Thus, the efficiency of MH therapy needs enhancement. In its recent 60-year of history, the field of MH has focused primarily on heating using MNPs for therapeutic applications. Increasing the thermal conversion efficiency of MNPs is the fundamental strategy for improving therapeutic efficacy. Recently, emerging experimental evidence indicates that MNPs-MH produces nano-scale heat effects without macroscopic temperature rise. A deep understanding of the effect of this localized induction heat for the destruction of subcellular/cellular structures further supports the efficacy of MH in improving therapeutic therapy. In this review, the currently available strategies for improving the antitumor therapeutic efficacy of MNPs-MH will be discussed. Firstly, the recent advancements in engineering MNP size, composition, shape, and surface to significantly improve their energy dissipation rates will be explored. Secondly, the latest studies depicting the effect of local induction heat for selectively disrupting cells/intracellular structures will be examined. Thirdly, strategies to enhance the therapeutics by combining MH therapy with chemotherapy, radiotherapy, immunotherapy, photothermal/photodynamic therapy (PDT), and gene therapy will be reviewed. Lastly, the prospect and significant challenges in MH-based antitumor therapy will be discussed. This review is to provide a comprehensive understanding of MH for improving antitumor therapeutic efficacy, which would be of utmost benefit towards guiding the users and for the future development of MNPs-MH towards successful application in medicine.

Graphene Oxide-Grafted Magnetic Nanorings Mediated Magnetothermodynamic Therapy Favoring Reactive Oxygen Species-Related Immune Response for Enhanced Antitumor Efficacy.

In this study, a magnetothermodynamic (MTD) therapy is introduced as an efficient systemic cancer treatment, by combining the magnetothermal effect and the reactive oxygen species (ROS)-related immunologic effect, in order to overcome the obstacle of limited therapeutic efficacy in current magnetothermal therapy (MTT). This approach was achieved by the development of an elaborate ferrimagnetic vortex-domain iron oxide nanoring and graphene oxide (FVIOs-GO) hybrid nanoparticle as the efficient MTD agent. Such a FVIOs-GO nanoplatform was shown to have high thermal conversion efficiency, and it was further proved to generate a significantly amplified ROS level under an alternating magnetic field (AMF). Both in vitro and in vivo results revealed that amplified ROS generation was the dominant factor in provoking a strong immune response at a physiological tolerable temperature below 40 °C in a hypoxic tumor microenvironment. This was supported by the exposure of calreticulin (CRT) on 83% of the 4T1 breast cancer cell surface, direct promotion of macrophage polarization to pro-inflammatory M1 phenotypes, and further elevation of tumor-infiltrating T lymphocytes. As a result of the dual action of magnetothermal effect and ROS-related immunologic effect, impressive in vivo systemic therapeutic efficacy was attained at a low dosage of 3 mg Fe/kg with two AMF treatments, as compared to that of MTT (high dosage of 6-18 mg/kg under four to eight AMF treatments). The MTD therapy reported here has highlighted the inadequacy of conventional MTT that solely relies on the heating effect of the MNPs. Thus, by employing a ROS-mediated immunologic effect, future cancer magnetotherapies can be designed with greatly improved antitumor capabilities.

Proton-driven transformable nanovaccine for cancer immunotherapy.

Cancer vaccines hold great promise for improved cancer treatment. However, endosomal trapping and low immunogenicity of tumour antigens usually limit the efficiency of vaccination strategies. Here, we present a proton-driven nanotransformer-based vaccine, comprising a polymer-peptide conjugate-based nanotransformer and loaded antigenic peptide. The nanotransformer-based vaccine induces a strong immune response without substantial systemic toxicity. In the acidic endosomal environment, the nanotransformer-based vaccine undergoes a dramatic morphological change from nanospheres (about 100 nanometres in diameter) into nanosheets (several micrometres in length or width), which mechanically disrupts the endosomal membrane and directly delivers the antigenic peptide into the cytoplasm. The re-assembled nanosheets also boost tumour immunity via activation of specific inflammation pathways. The nanotransformer-based vaccine effectively inhibits tumour growth in the B16F10-OVA and human papilloma virus-E6/E7 tumour models in mice. Moreover, combining the nanotransformer-based vaccine with anti-PD-L1 antibodies results in over 83 days of survival and in about half of the mice produces complete tumour regression in the B16F10 model. This proton-driven transformable nanovaccine offers a robust and safe strategy for cancer immunotherapy.

Near-Infrared Light Irradiation Induced Mild Hyperthermia Enhances Glutathione Depletion and DNA Interstrand Cross-Link Formation for Efficient Chemotherapy.

DNA alkylating agents generally kill tumor cells by covalently binding with DNA to form interstrand or intrastrand cross-links. However, in the case of cisplatin, only a few DNA adducts (<1%) are highly toxic irreparable interstrand cross-links. Furthermore, cisplatin is rapidly detoxified by high levels of intracellular thiols such as glutathione (GSH). Since the discovery of its mechanism of action, people have been looking for ways to directly and efficiently remove intracellular GSH and increase interstrand cross-links to improve drug efficacy and overcome resistance, but there has been little breakthrough. Herein, we hypothesized that the anticancer efficiency of cisplatin can be enhanced through iodo-thiol click chemistry mediated GSH depletion and increased formation of DNA interstrand cross-links via mild hyperthermia triggered by near-infrared (NIR) light. This was achieved by preparing an amphiphilic polymer with platinum(IV) (Pt(IV)) prodrugs and pendant iodine atoms (iodides). The polymer was further used to encapsulate IR780 and assembled into Pt-I-IR780 nanoparticles. Induction of mild hyperthermia (43 °C) at the tumor site by NIR light irradiation had three effects: (1) it accelerated the GSH-mediated reduction of Pt(IV) in the polymer main chain to platinum(II) (Pt(II)); (2) it boosted the iodo-thiol substitution click reaction between GSH and iodide, thereby attenuating the GSH-mediated detoxification of cisplatin; (3) it increased the proportion of highly toxic and irreparable Pt-DNA interstrand cross-links. Therefore, we find that mild hyperthermia induced via NIR irradiation can enhance the killing of cancer cells and reduce the tumor burden, thus delivering efficient chemotherapy.

Drug Delivery Based on Nanotechnology for Target Bone Disease.

Bone diseases are a serious problem in modern human life. With the coming acceleration of global population ageing, this problem will become more and more serious. Due to the specific physiological characteristics and local microenvironment of bone tissue, it is difficult to deliver drugs to the lesion site. Therefore, the traditional orthopedic medicine scheme has the disadvantages of high drug frequency, large dose and relatively strong side effects. How to target deliver drugs to the bone tissue or even target cells is the focus of the development of new drugs. Nano drug delivery system with a targeting group can realize precise delivery of orthopedic drugs and effectively reduce the systemic toxicity. In addition, the application of bone tissue engineering scaffolds and biomedical materials to realize in situ drug delivery also are research hotspot. In this article, we briefly review the application of nanotechnology in targeted therapies for bone diseases.

Self-Supply of O2 and H2O2 by a Nanocatalytic Medicine to Enhance Combined Chemo/Chemodynamic Therapy.

Combined chemo/chemodynamic therapy is a promising strategy to achieve an improved anticancer effect. However, the hypoxic microenvironment and limited amount of H2O2 in most solid tumors severely restrict the efficacy of this treatment. Herein, the construction of a nanocatalytic medicine, CaO2@DOX@ZIF-67, via a bottom-up approach is described. CaO2@DOX@ZIF-67 simultaneously supplies O2 and H2O2 to achieve improved chemo/chemodynamic therapy. In the weakly acidic environment within tumors, CaO2@DOX@ZIF-67 is broken down to rapidly release the Fenton-like catalyst Co2+ and the chemotherapy drug doxorubicin (DOX). The unprotected CaO2 reacts with H2O to generate both O2 and H2O2. The generated O2 relieves the hypoxia in the tumor and further improve the efficacy of DOX. Meanwhile, the generated H2O2 reacts with Co2+ ions to produce highly toxic •OH through a Fenton-like reaction, resulting in improved chemodynamic therapy.

Magnetic Nanomaterials for Advanced Regenerative Medicine: The Promise and Challenges.

The recent emergence of numerous nanotechnologies is expected to facilitate the development of regenerative medicine, which is a tissue regeneration technique based on the replacement/repair of diseased tissue or organs to restore the function of lost, damaged, and aging cells in the human body. In particular, the unique magnetic properties and specific dimensions of magnetic nanomaterials make them promising innovative components capable of significantly advancing the field of tissue regeneration. Their potential applications in tissue regeneration are the focus here, beginning with the fundamentals of magnetic nanomaterials. How nanomaterials-both those that are intrinsically magnetic and those that respond to an externally applied magnetic field-can enhance the efficiency of tissue regeneration is also described. Applications including magnetically controlled cargo delivery and release, real-time visualization and tracking of transplanted cells, magnetic regulation of cell proliferation/differentiation, and magnetic activation of targeted ion channels and signal pathways involved in regeneration are highlighted, and comments on the perspectives and challenges in magnetic nanomaterial-based tissue regeneration are given.

Regulation of Ca2+ Signaling for Drug-Resistant Breast Cancer Therapy with Mesoporous Silica Nanocapsule Encapsulated Doxorubicin/siRNA Cocktail.

Multidrug resistance (MDR) is the key cause that accounts for the failure of clinical cancer chemotherapy. To address the problem, herein, we presented an alternative strategy to conquer drug-resistant breast cancer through the combinatorial delivery of Ca2+ channel siRNA with cytotoxic drugs. Mesoporous silica nanocapsules (MSNCs) with mesoporous and hollow structure were fabricated for co-delivery of T-type Ca2+ channel siRNA and doxorubicin (DOX) with high drug loading efficiency. The DOX/siRNA co-loaded MSNCs showed a synergistic therapeutic effect on drug-resistant breast cancer cells MCF-7/ADR, while had only an additive effect on the drug-sensitive MCF-7 counterpart. It was found that the combination of T-type Ca2+ channel siRNA and DOX had a similar effect on MCF-7 and MCF-7/ADR in the knockdown of overexpressed T-type Ca2+ channels and decrease in cytosolic Ca2+ concentration ([Ca2+]i), but it specifically induced G0/G1 phase cell-cycle arrest and intracellular drug accumulation enhancement in MCF-7/ADR. The in vitro and in vivo results demonstrated that the MSNCs with good biocompatibility had a high efficiency for conquering the drug-resistant breast cancer with the DOX/calcium channel siRNA cocktail co-delivery. It provides a biological target for drug/gene delivery enhanced cancer therapy with nanoformulations.

Bioreducible Zinc(II)-Dipicolylamine Functionalized Hyaluronic Acid Mediates Safe siRNA Delivery and Effective Glioblastoma RNAi Therapy.

RNA interference (RNAi) is an emerging therapeutic modality for tumors. However, lack of a safe and efficient small interfering RNA (siRNA) delivery system limits its clinical application. Here, we report a bioreducible and less-cationic siRNA delivery carrier by conjugating Zn(II)-dipicolylamine complexes (Zn-DPA) onto hyaluronic acid (HA) via a redox-sensitive disulfide (-SS-) linker. Such polymer conjugates can formulate stable siRNA nanomedicines via coordination between zinc ions of DPA and the anionic phosphate of siRNA. After the conjugates are taken up by cells, intracellular reduction stimulus subsequently triggers the release of siRNAs and elucidates the desired RNAi effect. Our studies showed the formulated siRNA nanomedicines can be efficiently delivered into tumor cells/tissues and mediates less cytotoxicities both in vitro and in vivo. More importantly, when applied in a xenograft glioblastoma tumor model, this siRNA nanomedicine demonstrated significantly enhanced antitumor ability comparing to naked siRNA. This work demonstrates that such bioreducible Zn-DPA-functionalized HA conjugates without using cationic material as a siRNA carrier represents a promising direction for RNAi-based cancer therapy.

Enhanced Radiosensitization by Gold Nanoparticles with Acid-Triggered Aggregation in Cancer Radiotherapy.

An ideal radiosensitizer holding an enhanced tumor retention can play an incredible role in enhancing tumor radiotherapy. Herein, a strategy of acid-triggered aggregation of small-sized gold nanoparticles (GNPs) system within tumor is proposed and the resulting GNPs aggregates are applied as a radiosensitizer in vitro and in vivo. The GNPs system with the acid-triggered aggregation achieves an enhanced GNPs accumulation and retention in cancer cells and tumors in the form of the resulted GNPs aggregates. As a consequence, the radiosensitization effect shows significant improvement in cancer radiotherapy, which is shown in the studies of DNA breakage and the comet assay, and the sensitizer enhancement ratio (SER) value of the GNPs system (1.730) with MCF-7 cancer cells is much larger than that of the single GNPs (1.16). In vivo antitumor studies reveal that the GNPs system also enhances the sensitivity of MCF-7 tumor xenograft to radiotherapy. Furthermore, the GNPs aggregates improve the signal of small GNPs in vivo photoacoustic imaging. This study provides a new strategy and insights into fabricating nanoaggregates to magnify the radiosensitive efficiency of nanosystems in cancer radiotherapy.

Carbon-dot-supported atomically dispersed gold as a mitochondrial oxidative stress amplifier for cancer treatment.

Mitochondrial redox homeostasis, the balance between reactive oxygen species and antioxidants such as glutathione, plays critical roles in many biological processes, including biosynthesis and apoptosis, and thus is a potential target for cancer treatment. Here, we report a mitochondrial oxidative stress amplifier, MitoCAT-g, which consists of carbon-dot-supported atomically dispersed gold (CAT-g) with further surface modifications of triphenylphosphine and cinnamaldehyde. We find that the MitoCAT-g particles specifically target mitochondria and deplete mitochondrial glutathione with atomic economy, thus amplifying the reactive oxygen species damage caused by cinnamaldehyde and finally leading to apoptosis in cancer cells. We show that imaging-guided interventional injection of these particles potently inhibits tumour growth in subcutaneous and orthotopic patient-derived xenograft hepatocellular carcinoma models without adverse effects. Our study demonstrates that MitoCAT-g amplifies the oxidative stress in mitochondria and suppresses tumour growth in vivo, representing a promising agent for anticancer applications.

Defect-related luminescent bur-like hydroxyapatite microspheres induced apoptosis of MC3T3-E1 cells by lysosomal and mitochondrial pathways.

When orthopedic joints coated by hydroxyapatite (HA) were implanted in the human body, they release wear debris into the surrounding tissues. The generation and accumulation of wear particles will induce aseptic loosening. However, the potential bioeffect and mechanism of HA-coated orthopedic implants on bone cells are poorly understood. In this study, defect-related luminescent bur-like hydroxyapatite (BHA) microspheres with the average diameter of 7-9 µm which are comparable to that of the wear-debris particles from aseptically loosened HA implants or HA debris have been synthesized by hydrothermal synthesis and the MC3T3-E1 cells were set as a cells model to study the potential bioeffect and mechanism of BHA microspheres. The studies demonstrated that BHA microspheres could be taken into MC3T3-E1 cells via endocytosis involved in micropinocytosis- and clathrin-mediated endocytosis process, and exert cytotoxicity effect. BHA microspheres could induce the cell apoptosis by intracellular production of reactive oxygen species (ROS), which led to not only an increase in the permeability of lysosome and release of cathepsins B, but also mitochondrial dysfunction and DNA damage. Our results provide novel evidence to elucidate their toxicity mechanisms and might be helpful for more reasonable applications of HA-based orthopaedic implants in the future.