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.

In Vivo Immune Adjuvant Effects of CaCO3 Nanoparticles through Intracellular Ca2+ Concentration Regulation.

Calcium (Ca) is a vital component of the human body and plays a crucial role in intracellular signaling and regulation as a second messenger. Recent studies have shown that changes in intracellular Ca2+ concentration can influence immune cell function. In this study, we developed calcium carbonate nanoparticles (CaNPs) of various sizes using a Nanosystem Platform to modulate intracellular Ca2+ concentration in vitro and in vivo. Our findings demonstrate that intravenous administration of CaNPs led to changes in the number and ratio of immune cells in the spleen and stimulated the activation of dendritic cells (DCs) and macrophages. Notably, CaNPs exhibited strong adjuvant properties in the absence of antigenic stimuli. These results indicate that CaNPs have the potential to regulate immune cell function by modulating Ca2+ concentrations, offering a novel approach for disease prevention and treatment in combination with antigens or drugs. Overall, our study emphasizes the importance of modulating intracellular Ca2+ concentration as a means of regulating immune cell function.

Co-delivery of vitamin D3 and Lkb1 siRNA by cationic lipid-assisted PEG-PLGA nanoparticles to effectively remodel the immune system in vivo.

The imbalance of the immune system can lead to the occurrence of autoimmune diseases. Controlling and regulating the proliferation and function of effector T (Teff) cells and regulatory T (Treg) cells becomes the key to treating these diseases. Dendritic cells (DCs), as dedicated antigen-presenting cells, play a key role in inducing the differentiation of naive CD4+ T cells. In this study, we designed a cationic lipid-assisted PEG-PLGA nanoparticle (NPs/VD3/siLkb1) to deliver 1,25-dihydroxyvitamin D3 (VD3) and small interfering RNA (siRNA) to DC cells in the draining lymph nodes. By modulating the phenotypic changes of DC cells, this approach expands Treg cells and reduces the occurrence of autoimmune diseases. Thus, this study provides a novel approach to alleviating the occurrence and development of autoimmune diseases while also minimizing the risk of unwanted complications.

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.

Nanoparticle delivery of CD40 siRNA suppresses alloimmune responses by inhibiting activation and differentiation of DCs and macrophages.

CD40 is an important costimulatory molecule expressed on antigen-presenting cells (APCs) and plays a critical role for APC activation, offering a promising therapeutic target for preventing allograft rejection. Here, we developed a biodegradable nanoparticle small interfering RNA delivery system (siCD40/NPs) to effectively deliver CD40 siRNA (siCD40) into hematopoietic stem cells (HSCs), myeloid progenitors, and mature dendritic cells (DCs) and macrophages. Injection of siCD40/NPs not only down-regulated CD40 expression in DCs and macrophages but also inhibited the differentiation of HSCs and/or myeloid progenitors into functional DCs and macrophages. Furthermore, siCD40/NPs treatment significantly prolonged allograft survival in mouse models of skin allotransplantation. In addition to reiteration of the role of CD40 in APC activation, our findings highlight a previously unappreciated role of CD40 in DC and macrophage differentiation from their progenitors. Furthermore, our results support the effectiveness of siCD40/NPs in suppressing alloimmune responses, providing a potential means of facilitating tolerance induction and preventing allotransplant rejection.

Lipid-mediated delivery of CD47 siRNA aids JQ1 in ensuring simultaneous downregulation of PD-L1 and CD47 and improves antitumor immunotherapy efficacy.

Cancer immunotherapy using immune checkpoint blockade has become an attractive treatment option for patients with different cancers. JQ1, an indirect inhibitor of MYC, enhances antitumor immune responses by regulating the expression of programmed death-ligand 1 (PD-L1) and cluster of differentiation 47 (CD47) in tumor cells; however, its role in downregulating the expression of CD47 remains elusive. The present study revealed that JQ1 failed to downregulate and, when used at high concentrations, it unexpectedly upregulated the expression of CD47 in murine B16F10 melanoma and 4T1 breast tumor cells. Hence, the combinatory use of JQ1 and CD47-specific short interfering RNA (siRNA) may lead to an improved antitumor effect. To overcome the poor water solubility of JQ1 and enhance tumor-targeted delivery, cationic lipid nanoparticles (CLNs) encapsulating both JQ1 and siCD47 simultaneously (CLN/JQ1/siCD47) or each individually (CLN/JQ1/siNC or CLN/siCD47) were prepared. CLN/JQ1/siCD47, but not CLN/JQ1/siNC or CLN/siCD47, simultaneously downregulated both PD-L1 and CD47 in vitro and in vivo. Furthermore, compared with CLN/JQ1/siNC and CLN/siCD47, CLN/JQ1/siCD47 induced a significantly enhanced antitumor effect in mice with established breast cancer. The results of this study highlight a synergistic effect of simultaneous PD-L1 and CD47 downregulation and provide a novel strategy for improving the antitumor effects of JQ1.

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.

Kidney functional stages influence the role of PEG end-group on the renal accumulation and distribution of PEGylated nanoparticles.

Modification with polyethylene glycol (PEG), or PEGylation, has become a popular method to improve the efficiency of drug delivery in vivo using nanoparticle-based delivery systems. The PEG end-group plays an important role in the in vivo fate of PEGylated nanoparticles through its interactions with proteins in the serum and the cell membrane. However, the effects of PEG end-groups on the renal clearance of PEGylated nanoparticles remain unclear. Kidney function may also affect the renal accumulation and distribution of nanoparticles. Herein, we demonstrate that the accumulation and distribution of PEGylated nanoparticles in kidneys are significantly affected by both the PEG end-group and kidney function damage. Interestingly, compared to PEG with an amino or methoxy end-group, PEG with maleimide as the end-group markedly enhanced the accumulation of PEGylated nanoparticles in normal kidneys, which may improve renal clearance. However, obvious enhancements in the renal accumulation and medullary distribution of PEGylated nanoparticles are detected in kidneys with functional impairment. Damage to renal function further affects how the PEG end-group influences the accumulation and distribution of PEGylated nanoparticles in kidneys in vivo. Collectively, the findings provide deep insights into the interactions between PEGylated nanoparticles and kidneys in vivo.

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.

Photodynamic therapy produces enhanced efficacy of antitumor immunotherapy by simultaneously inducing intratumoral release of sorafenib.

Photodynamic therapy (PDT) can destroy local tumor cells and induce effective antitumor immune responses, and has been applied in the treatment of patients with superficial solid tumors. Numerous systemic side effects of PDT, such as pain and skin photosensitivity, however, limit this therapeutic option. In addition, the immunosuppressive tumor microenvironment has been found to be another critical barrier for the antitumor immunity induced by PDT. Therefore, effectively enhancing the cytotoxicity to tumor cells of low-dose PDT and inhibiting the tumor immunosuppressive tumor microenvironment may be a feasible strategy to overcome these drawbacks of PDT. Here, a sorafenib and chlorin e6 co-loaded reactive oxygen species (ROS)-responsive nanoparticle (NP-sfb/ce6) is developed to improve antitumor responses by intratumoral release of sorafenib at the time of PDT. Under 660-nm laser irradiation, ROS produced by chlorin e6 (ce6) destruct the nanoparticles, resulting in boosted sorafenib cascade release. The rapidly released sorafenib acts synergistically with the low-dose PDT to inhibit tumor growth by inducing strong T cell-dependent local and systemic antitumor immune responses, reprograming the tumor immune microenvironment, and limiting the interaction between cytotoxic CD8+ T cells and immunosuppressive cells. This study provides new avenues for cascade-amplifying antitumor effects of photodynamic therapy.

Intratumoral delivery of M-CSF by calcium crosslinked polymer micelles enhances cancer immunotherapy.

Immunotherapy has shown promising results in multiple malignancies. However, there are still significant challenges in cancer immunotherapy including the powerful immunosuppressive tumor microenvironment and adverse off-target side effects. Nanomaterials with defined physico-biochemical properties are versatile drug delivery platforms that may address these key technical challenges faced by cancer immunotherapy. Here, a tumor acidity-responsive biomacromolecule delivery system was designed to intratumorally deliver an immune-activating cytokine, macrophage colony-stimulating factor (M-CSF) and attenuate the acidic microenvironment. This nanoparticle was prepared by introducing CaCO3 as a crosslinker to form an M-CSF-loaded stable micelle (NP/M-CSF/CaCO3). Administration of NP/M-CSF/CaCO3 significantly inhibited tumor growth by enhancing T cell-mediated anti-tumor immune responses and reversing the TAM-mediated immunosuppression. This study provides new avenues for cascade amplification of the antitumor effects by targeting the tumor microenvironment. This approach may also help avoid unwanted complications.

The influence of tumor-induced immune dysfunction on the immune cell distribution of gold nanoparticles in vivo.

Gold nanoparticles (AuNPs) have been extensively explored as a drug carrier and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy for cancer. Although the mononuclear phagocyte system and immune system are known to play the main roles in the clearance of AuNPs during the circulation, the particle distribution within the immune cells under the condition of immune dysfunction caused by tumor growth has not been thoroughly studied. Here, the cellular distribution of Cy5 labeled AuNPs with diameters of 5, 30 and 50 nm is characterized within the immune populations of the blood, spleen and bone marrow from tumor free and tumor bearing mice using flow cytometry. Tumor-associated immune dysfunction was observed in all immune organs and cell lineages, and it changed with tumor growth. Furthermore, the particle cellular distribution significantly changed in the tumor bearing mice compared with the tumor free mice. Finally, the particle distribution in the immune cells was also different at different stages of the tumor. Overall, these results can help inform and influence future AuNP design criteria including the future applications for nanoparticle-mediated cancer therapy.