Targeted delivery strategies: The interactions and applications of nanoparticles in liver diseases.

In recent years, nanoparticles have been broadly utilized in various drugs delivery formulations. Nanodelivery systems have shown promise in solving problems associated with the distribution of hydrophobic drugs and have promoted the accumulation of nanomedicines in the circulation or in organs. However, the injection dose of nanoparticles (NPs) is much greater than that needed by diseased tissues or organs. In other words, most of the NPs are localized off-target and do not reach the desired tissue or organs. With the rapid development of biodegradable and biosafety nanomaterials, the nanovectors represent assurance of safety. However, the off-target effects also induce concerns about the application of NPs, especially in the delivery of gene editing tools. Therefore, a complete understanding of the biological responses to NPs in the body will clearly guide the design of targeted delivery of NPs. The different properties of various nanodelivery systems may induce diverse interactions between carriers and organs. In this review, we describe the relationship between the liver, the most influenced organ of systemic administration of NPs, and targeted delivery nanoplatforms. Various transport vehicles have adopted multiple delivery strategies for the targeted delivery to the cells in the homeostasis liver and in diseased liver. Additionally, nanodelivery systems provide a novel strategy for treating incurable diseases. The appearance of a targeted delivery has profoundly improved the application of NPs to liver diseases.

Expression of a mutant CD47 protects against phagocytosis without inducing cell death or inhibiting angiogenesis.

CD47 is a ligand of SIRPα, an inhibitory receptor expressed by macrophages, dendritic cells, and natural killer (NK) cells, and, therefore, transgenic overexpression of CD47 is considered an effective approach to inhibiting transplant rejection. However, the detrimental effect of CD47 signaling is overlooked when exploring this approach. Here, we construct a mutant CD47 by replacing the transmembrane and intracellular domains with a membrane anchor (CD47-IgV). In both human and mouse cells, CD47-IgV is efficiently expressed on the cell surface and protects against phagocytosis in vitro and in vivo but does not induce cell death or inhibit angiogenesis. Furthermore, hematopoietic stem cells expressing transgenic CD47-IgV show no detectable alterations in engraftment or differentiation. This study provides a potentially effective means of achieving transgenic CD47 expression that may help to produce gene-edited pigs for xenotransplantation and hypoimmunogenic pluripotent stem cells for regenerative medicine.

Impacts of cationic lipid-DNA complexes on immune cells and hematopoietic cells in vivo.

The inability to systemic administration of nanoparticles, particularly cationic nanoparticles, has been a significant barrier to their clinical translation due to toxicity concerns. Understanding the in vivo behavior of cationic lipids is crucial, given their potential impact on critical biological components such as immune cells and hematopoietic stem cells (HSC). These cells are essential for maintaining the body's homeostasis, and their interaction with cationic lipids is a key factor in determining the safety and efficacy of these nanoparticles. In this study, we focused on the cytotoxic effects of cationic lipid/DNA complexes (CLN/DNA). Significantly, we observed that the most substantial cytotoxic effects, including a marked increase in numbers of long-term hematopoietic stem cells (LT-HSC), occurred 24 h post-CLN/DNA treatment in mice. Furthermore, we found that CLN/DNA-induced HSC expansion in bone marrow (BM) led to a notable decrease in the ability to reestablish blood cell production. Our study provides crucial insights into the interaction between cationic lipids and vital cellular components of the immune and hematopoietic systems.

Maleimide as the PEG end-group promotes macrophage-targeted drug delivery of PEGylated nanoparticles in vivo by enhancing interaction with circulating erythrocytes.

Radiotherapy (IR) is capable of enhancing antitumor immune responses. However, IR treatment also aggravates the infiltration of peripheral macrophages into the tumor, resulting in reversing the therapeutic effects of antitumor immunity. Thus, a strategy to effectively prevent tumor infiltration by macrophages may further improved the therapeutic efficacy of radiotherapy. Herein, we found that PEGylated solid lipid nanoparticles with maleimide as PEG end-group (SLN-PEG-Mal) show significantly enhanced adsorption onto RBCs through reacting with reactive sulfhydryl groups on RBCs' surface both in vitro and in vivo, and caused significant changes in the surface properties and morphology of RBCs. These RBCs adsorbed by SLN-PEG-Mal were rapidly removed from circulation due to efficient engulfment by reticuloendothelial macrophages, supporting the usefulness of SLN-PEG-Mal for macrophage-targeted drug delivery. While lacking the use of radioisotope tracing (considered the gold standard for PK/BD studies), our data align with the expected pathway of host defense activation through surface-loaded RBCs. Importantly, injection of paclitaxel-loaded SLN-PEG-Mal effectively inhibited the tumor-infiltration by macrophages, and significantly improved the antitumor immune responses in tumor-bearing mice treated with low-dose irradiation. This study provides insights into the effects of maleimide as PEG end-group on enhancing the interaction between PEGylated nanoparticles and RBCs and offers an effective strategy to inhibit tumor infiltration by circulating macrophages.

Biosafety risk assessment of gold and aluminum nanoparticles in tumor-bearing mice.

To improve the biosafety of the nanodelivery system, this study developed novel monodisperse spherical aluminum nanoparticles (Al NPs) and evaluated their cytotoxicity in vitro and distribution and biotoxicity in vivo. Compared with gold nanoparticles of the same size, Al NPs not only had low cytotoxicity in vitro but also did not cause accumulation in major organs in vivo after intravenous injections. No significant abnormalities were observed in the serum biochemical indices of mice injected with Al NPs. Additionally, no substantial changes occurred in the histopathology of major organs, and no apparent biological toxicity was measured after consecutive injections of Al NPs. These results indicate that Al NPs have a good biological safety and provide a new method for developing low-toxicity nanomedicine.

Vibratome sectioning of tumors to evaluate the interactions between nanoparticles and the tumor microenvironment ex-vivo.

Nanoparticles have been investigated as drug carriers and promising agents for cancer therapy. However, the tumor microenvironment (TME), which is formed by the tumor, is considered a barrier for nanocarriers to enter the internal tumor tissue. Therefore, the evaluation of the biological distribution of nanocarriers in TME can provide useful information on their role in tumor-targeted drug delivery. Although the tumor-bearing mouse model is commonly used to investigate the distribution of nanocarriers in the TME, there is currently a lack of a testing system to predict the distribution of nanocarriers in tumor tissues, especially in patients. This study revealed that the macrophages and dendritic cells (DCs) were more distributed in the peripheral part than the central part of the tumor, which might be an obstacle to the uniform distribution of nanoparticles in the tumor. In addition, the cellular uptake of gold nanoparticles (AuNR and AuNS) in macrophages and DCs cell lines (RAW264.7 and DC1.2) was markedly different from that in the TME. Hence, the study model of the interaction between nanoparticles and macrophages and DCs has an important impact on the accuracy of the results. The vibratome sections of tumor tissues preserved the spatial distribution of immune cells and tumor cells, and had very little effects on their morphologies and activities. More importantly, we found that the distribution of nanocarriers in vibratome sections was similar to that in tumors in vivo. In all, ex vivo analysis using vibratome sections of tumor tissues provides a more convenient and stable method for elucidating the influences of TME on the distribution of nanocarriers.

An optimized ionizable cationic lipid for brain tumor-targeted siRNA delivery and glioblastoma immunotherapy.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a high mortality rate. Immunotherapy has achieved promising clinical results in multiple cancers, but shows unsatisfactory outcome in GBM patients, and poor drug delivery across the blood-brain barrier (BBB) is believed to be one of the main limitations that hinder the therapeutic efficacy of drugs. Herein, a new cationic lipid nanoparticle (LNP) that can efficiently deliver siRNA across BBB and target mouse brain is prepared for modulating the tumor microenvironment for GBM immunotherapy. By designing and screening cationic LNPs with different ionizable amine headgroups, a lipid (named as BAMPA-O16B) is identified with an optimal acid dissociation constant (pKa) that significantly enhances the cellular uptake and endosomal escape of siRNA lipoplex in mouse GBM cells. Importantly, BAMPA-O16B/siRNA lipoplex is highly effective to deliver siRNA against CD47 and PD-L1 across the BBB into cranial GBM in mice, and downregulate target gene expression in the tumor, resulting in synergistically activating a T cell-dependent antitumor immunity in orthotopic GBM. Collectively, this study offers an effective strategy for brain targeted siRNA delivery and gene silencing by optimizing the physicochemical property of LNPs. The effectiveness of modulating immune environment of GBM could further be expanded for potential treatment of other brain tumors.

A biocompatible nanoparticle-based approach to inhibiting renal ischemia reperfusion injury in mice by blocking thrombospondin-1 activity.

Thrombospondin-1 (TSP-1) is a key mediator of renal ischemia-reperfusion injury (IRI), a major cause of kidney dysfunction under various disease conditions and a risk factor of renal allograft rejection. In this study, we developed a nanotechnology-based therapy targeting TSP-1 to prevent renal IRI. A biocompatible nanoparticle (NP) capable of specific binding to TSP-1 was prepared by conjugating NPs with TSP-1-binding (LSKL) peptides. LSKL/NPs not only effectively adsorbed recombinant TSP-1 proteins in vitro, but also efficiently neutralized TSP-1 in mice undergoing renal IRI. IRI-induced elevation of TSP-1 in the kidney was significantly inhibited by post-IR treatment with LSKL/NPs, but not free LSKL or NPs. Furthermore, TSP-1 proteins adsorbed on LSKL/NPs were functionally inactive and unable to induce apoptosis in renal tubular epithelial cells. Importantly, LSKL/NPs induced strong protection against renal IRI, as shown by markedly diminished serum creatinine levels and improved histological lesions of the kidney. Thus, LSKL/NPs provide a useful means of depleting and inactivating TSP-1 and a potential therapy for renal IRI.

Posttransplant blockade of CXCR4 improves leukemia complete remission rates and donor stem cell engraftment without aggravating GVHD.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a promising therapeutic option for hematological malignancies, but relapse resulting predominantly from residual disease in the bone marrow (BM) remains the major cause of treatment failure. Using immunodeficient mice grafted with laboratory-generated human B-ALL, our previous study suggested that leukemia cells within the BM are resistant to graft-versus-leukemia (GVL) effects and that mobilization with CXCR4 antagonists may dislodge leukemia cells from the BM, enabling them to be destroyed by GVL effects. In this study, we extended this approach to patient-derived xenograft (PDX) and murine T-ALL and AML models to determine its clinical relevance and effects on GVHD and donor hematopoietic engraftment. We found that posttransplant treatment with the CXCR4 antagonist AMD3100 significantly improved the eradication of leukemia cells in the BM in PDX mice grafted with B-ALL cells from multiple patients. AMD3100 also significantly improved GVL effects in murine T-ALL and AML models and promoted donor hematopoietic engraftment in mice following nonmyeloablative allo-HCT. Furthermore, posttransplant treatment with AMD3100 had no detectable deleterious effect related to acute or chronic GVHD. These findings provide important preclinical data supporting the initiation of clinical trials exploring combination therapy with CXCR4 antagonists and allo-HCT.

The Host-Defense-Peptide-Mimicking Synthetic Polypeptides Effectively Enhance Antitumor Immunity through Promoting Immunogenic Tumor Cell Death.

Cancer immunotherapy is to artificially stimulate the immune system against tumor cells. Effectively increasing the immunogenicity of dying tumor cells has great potential to stimulate the anticancer immune responses. Recently, a synthetic cationic anticancer polypeptide (ACPP) is prepared, which mimics the host defense peptides, to effectively inhibit tumor growth by directly inducing rapid necrosis of cancer cells through a membrane-lytic mechanism. Thus, this ACPP has the potential ability to induce immunogenic cancer cell death (ICD) and promote antitumor immunity. Herein, it is reported that ACPP successfully induces ICD in mouse colon cancer cells, resulting in effectively promoting T-cell-dependent antitumor immune responses by enhanced activation of dendritic cells. Interestingly, the level of natural killer cells, which are another kind of antitumor effector cell, in tumor microenvironment is also significantly increased by ACPP. The ratio of M1/M2 tumor-associated macrophages is further obviously increased, indicating that tumor immunosuppressive microenvironment has been effectively reprogramed. More importantly, it is found that the anticancer immunity induced by ACPP is dose dependent. Finally, 40% of the established CT26 tumors are completely eliminated by ACPP treatment with an optimized dose. This study proposes a simple and effective strategy for promoting cancer immunotherapy.

Cationic Liposome/DNA Complexes Mediate Antitumor Immunotherapy by Promoting Immunogenic Tumor Cell Death and Dendritic Cell Activation.

Immunotherapy has been successfully used in the treatment of multiple malignancies, but clinical studies revealed low response rates. Thus, the development of new effective immunotherapeutic modalities is urgently needed. Successfully inducing tumor cell death with enhanced antigenicity is important for the expansion and differentiation of tumor-specific CD8+ cytotoxic T lymphocytes. Cationic liposome/DNA complexes (CLN/DNA), which usually have obvious cytotoxic effects, may improve the antitumor immunity through enhancing the immunogenicity of dying tumor cells. Herein, we report that a plasmid DNA-encapsulated cationic lipid nanoparticle formulated with cholesterol, DOTAP, and DSPE-mPEG2000 significantly increases the tumor cell death with high antigenicity in vitro. Furthermore, the cationic liposome/DNA complex (CLN/DNA) treatment promotes the activation of dendritic cells (DCs). We also find that the intratumorally injected CLN/DNA successfully promoted the activation of DCs in the tumor-draining lymph node. Importantly, both local tumor growth and distant tumor formation were significantly inhibited by T cell-dependent antitumor immune responses after intratumoral injection of CLN/DNA. This study presents a simple and effective strategy for improving the cancer immunotherapy.

Intratumoral delivery of CCL25 enhances immunotherapy against triple-negative breast cancer by recruiting CCR9+ T cells.

CCR9+ T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9+ cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9+CD8+ T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell-dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein-1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration.

Fast and Efficient CRISPR/Cas9 Genome Editing In Vivo Enabled by Bioreducible Lipid and Messenger RNA Nanoparticles.

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.