Durable protective efficiency provide by mRNA vaccines require robust immune memory to antigens and weak immune memory to lipid nanoparticles.

The Pegylated lipids in lipid nanoparticle (LNPs) vaccines have been found to cause acute hypersensitivity reactions in recipients, and generate anti-LNPs immunity after repeated administration, thereby reducing vaccine effectiveness. To overcome these challenges, we developed a new type of LNPs vaccine (SAPC-LNPs) which was co-modified with sialic acid (SA) - lipid derivative and cleavable PEG - lipid derivative. This kind of mRNA vaccine can target dendritic cells (DCs) and rapidly escape from early endosomes (EE) and lysosomes with a total endosomal escape rate up to 98 %. Additionally, the PEG component in SAPC-LNPs was designed to detach from the LNPs under the catalysis of carboxylesterase in vivo, which reduced the probability of PEG being attached to LNPs entering antigen-presenting cells. Compared with commercially formulated vaccines (1.5PD-LNPs), mice treated with SAPC-LNPs generated a more robust immune memory to tumor antigens and a weaker immune memory response to LNPs, and showed lower side effects and long-lasting protective efficiency. We also discovered that the anti-tumor immune memory formed by SAPC-LNPs mRNA vaccine was directly involved in the immune cycle to rattack tumor. This immune memory continued to strengthen with multiple cycles, supporting that the immune memory should be incorporated into the theory of tumor immune cycle.

Sialic acid-modified doxorubicin liposomes target tumor-related immune cells to relieve multiple inhibitions of CD8+ T cells.

In different types of cancer treatments, cancer-specific T cells are required for effective anticancer immunity, which has a central role in cancer immunotherapy. However, due to the multiple inhibitions of CD8+ T cells by tumor-related immune cells, CD8+ T-cell mediated antitumor immunotherapy has not achieved breakthrough progress in the treatment of solid tumors. Receptors for sialic acid (SA) are highly expressed in tumor-associated immune cells, so SA-modified nanoparticles are a drug delivery nanoplatform using tumor-associated immune cells as vehicles. To relieve the multiple inhibitions of CD8+ T cells by tumor-associated immune cells, we prepared SA-modified doxorubicin liposomes (SL-DOX, Scheme 1A). In our study, free SA decreased the toxicity of SL-DOX to tumor-associated immune cells. Compared with common liposomes, SL-DOX could inhibit tumor growth more effectively. It is worth noting that SL-DOX could not only kill tumor-related neutrophils and monocytes to relieve the multiple inhibitions of CD8+ T cells but also induce immunogenic death of tumor cells to promote the infiltration and differentiation of CD8+ T cells (Scheme 1B). Therefore, SL-DOX has potential value for the clinical therapeutic effect of CD8+ T cells mediating anti-tumor immunotherapy.

Simultaneous dendritic cells targeting and effective endosomal escape enhance sialic acid-modified mRNA vaccine efficacy and reduce side effects.

mRNA vaccines are attractive prospects for the development of DC-targeted vaccines; however, no clinical success has been realized because, currently, it is difficult to simultaneously achieve DC targeting and efficient endosomal/lysosomal escape. Herein, we developed a sialic acid (SA)-modified mRNA vaccine that simultaneously achieved both. The SA modification promoted DCs uptake of lipid nanoparticles (LNPs) by 2 times, >90% of SA-modified LNPs rapidly escaped from early endosomes (EEs), avoided entering lysosomes, achieved mRNA simultaneously translated in ribosomes distributed in the cytoplasm and endoplasmic reticulum (ER), significantly improved the transfection efficiency of mRNA LNPs in DCs. Additionally, we applied cleavable PEG-lipids in mRNA vaccines for the first time and found this conducive to cellular uptake and DC targeting. In summary, SA-modified mRNA vaccines targeted DCs efficiently, and showed significantly higher EEs/lysosomal escape efficiency (90% vs 50%), superior tumor treatment effect, and lower side effects than commercially formulated mRNA vaccines.

Sialic acid-mediated photochemotherapy enhances infiltration of CD8+ T cells from tumor-draining lymph nodes into tumors of immunosenescent mice.

Immunoscenescence plays a key role in the initiation and development of tumors. Furthermore, immunoscenescence also impacts drug delivery and cancer therapeutic efficacy. To reduce the impact of immunosenescence on anti-tumor therapy, this experimental plan aimed to use neutrophils with tumor tropism properties to deliver sialic acid (SA)-modified liposomes into the tumor, kill tumor cells via SA-mediated photochemotherapy, enhance infiltration of neutrophils into the tumor, induce immunogenic death of tumor cells with chemotherapy, enhance infiltration of CD8+ T cells into the tumor-draining lymph nodes and tumors of immunosenescent mice, and achieve SA-mediated photochemotherapy. We found that CD8+ T cell and neutrophil levels in 16-month-old mice were significantly lower than those in 2- and 8-month-old mice; 16-month-old mice exhibited immunosenescence. The anti-tumor efficacy of SA-mediated non-photochemotherapy declined in 16-month-old mice, and tumors recurred after scabbing. SA-mediated photochemotherapy enhanced tumor infiltration by CD8+ T cells and neutrophils, induced crusting and regression of tumors in 8-month-old mice, inhibited metastasis and recurrence of tumors and eliminated the immunosenescence-induced decline in antitumor therapeutic efficacy in 16-month-old mice via the light-heat-chemical-immunity conversion.

Persistent immune response: Twice tumor exfoliation induced by sialic acid-modified vincristine sulfate liposomes.

Studies have shown that tumor-associated macrophages (TAMs) are crucial for the establishment and maintenance in immunosuppressive tumor immune microenvironment (TIME), which can help tumor cells to achieve immune escape and attenuate antitumor therapy. Siglecs, the receptors of sialic acid (SA), widely exist in TAMs, which could be targeted to disrupt TIME and inhibit tumor growth at the root. Therefore, a SA-modified VCR liposome was reported (VCR-SSAL). Cellular and pharmacodynamic experiments showed that VCR-SSAL exhibited strong TAMs targeting and tumor-killing ability. Interestingly, VCR-SSAL treatment induced a phenomenon in which the cancerous tissues were "fell off" from the growth site, after which the wound gradually healed. Three months after the wound healed, the mice whose tumors fell off were re-inoculated, and the tumor fell off again without treatment, with an exfoliation rate of 100%. We speculated that this special efficacy might be due to that VCR loaded in VCR-SSAL could activate adaptive immunity by inducing DNA damage, promoting cytotoxic T lymphocytes (CTLs) infiltration into tumor sites, and enhancing the antitumor immune response. Thus, this study might provide new insights into the application of traditional chemotherapeutic drugs.

The Fate of Sialic Acid and PEG Modified Epirubicin Liposomes in Aged versus Young Cells and Tumor Mice Models.

In preclinical studies of young mice, nanoparticles showed excellent anti-tumor therapeutic effects by harnessing Peripheral Blood Monocytes (PBMs) and evading the immune system. However, the changes of age will inevitably affect PBMs and the immune system, and there is a serious lack of relevant research. Sialic acid (SA)-octadecylamine (ODA) was synthesized, and SA- or polyethylene glycol (PEG)-modified epirubicin (EPI) liposomes (EPI-SL and EPI-PL, respectively) were prepared to explore differences in antitumor treatment using 8-month-old and 8-week-old Kunming mice. Based on presented data, 8-month-old mice had more PBMs in peripheral blood than 8-week-old mice, and age differences resulted in different anti-tumor treatment effects following EPI-SL and EPI-PL treatment. Following EPI-PL administration, the tumor volume was significantly smaller in 8-week-old mice than in 8-month-old mice (* p < 0.05). Eight-month-old mice treated with EPI-SL (8M-SL) presented no damage to healthy tissue, with a 100% survival rate, and 50% mice in 8M-SL showed 'shedding' of tumor tissues from the growth site. Accordingly, 8-month-old mice treated with EPI-SL achieved the best therapeutic effect at different ages and with different liposomes. EPI-SL could improve the antitumor effect of 8-week-old and 8-month-old mice.

Branched PEG-modification: A new strategy for nanocarriers to evade of the accelerated blood clearance phenomenon and enhance anti-tumor efficacy.

PEGylation is one of the most successful technologies for reducing immunogenicity, improving the stability and circulation time of nanocarriers, and has been applied in the clinic for over three decades. However, linear PEG-modified nanocarriers have been found to induce anti-PEG IgM at the first injection, which triggers the accelerated blood clearance (ABC) phenomenon upon repeated injections. Furthermore, clinical and research evidence has revealed that anti-PEG antibodies also cause serious complement activation-related pseudoallergies (CARPA), which greatly reduce the safety of linear PEGylated nanocarriers. In this study, as an alternative to linear PEG, branched PEG was selected owing to its low antigenicity. We pioneer the use of branched PEG lipid derivatives [DSPE-mPEG2,n (n = 2, 10, and 20 kDa)] to modify nanoemulsions (PE2,n) and liposomes (PL2,n). Upon characterization, PE2,n and PL2,n showed similar physicochemical properties to linear DSPE-mPEG2000-modified nanocarriers in terms of size, polydispersity index (PDI), and zeta potential. However, our pharmacokinetics study surprisingly indicated that PE2,n and PL2,n did not induce the ABC phenomenon after repeated injection. This may be attributed to the fact that PE2,n and PL2,n induced noticeably lower levels of anti-PEG IgM than linear PEG-modified nanocarriers and did not activate the complement system. Furthermore, we are the first to investigate the anti-tumor efficacy of DSPE-mPEG2,n-modified liposomal doxorubicin (DOX). The pharmacodynamic experiments showed that DSPE-mPEG2,n-m-modified liposomal DOX had better in vivo anti-tumor effects than linear DSPE-mPEG2000-modified liposomes. Therefore, we speculate that DSPE-mPEG2,n-modified nanocarriers possess promising prospects in avoiding the ABC phenomenon, reducing CARPA, and improving the anti-tumor efficacy of encapsulated drugs.

Sequential administration of sialic acid-modified liposomes as carriers for epirubicin and zoledronate elicit stronger antitumor effects with reduced toxicity.

Combined administration of drugs can improve efficacy and reduce toxicity; therefore, this combination approach has become a routine method in cancer therapy. The main combination regimens are sequential, mixed (also termed "cocktail"), and co-loaded; however, other combinations, such as administration of synergistic drugs and the use of formulations with different mechanisms of action, may exert better therapeutic effects. Tumor-associated macrophages (TAMs) play functional roles throughout tumor progression and exhibit characteristic phenotypic plasticity. Sialic acid (SA)-modified epirubicin liposomes (S-E-L) and SA-modified zoledronate liposomes (S-Z-L) administered separately kill TAMs, reverse their phenotype, and achieve antitumor effects. In this study, we examined the effects of a two-treatment combination for drug delivery, using sequential, mixed, and co-loaded drug delivery. We found that therapeutic effects differed between administration methods: mixed administration of S-E-L and S-Z-L, co-loaded administration of SA-modified liposomes (S-ZE-C), and sequential administration of S-E-L injected 24 h after S-Z-L did not inhibit tumor growth; however, sequential administration of S-Z-L injected 24 h after S-E-L resulted in no tumor growth, no toxicity to noncancerous tissue, and no death of mice, and exhibited 25% tumor shedding. Thus, our results thus encourage the further development of combined therapies for nanomedicines based on the mechanisms investigated here.

Influence of Dose on Neutrophil-Mediated Delivery of Nanoparticles for Tumor-Targeting Therapy Strategies.

It is well known that neutrophil-mediated delivery of therapeutic agents is a promising method for treating tumors. However, owing to the limited number and limited uptake ability of neutrophils, determining a reasonable dose has become an urgent problem to be solved. Furthermore, the number of nanoparticles is far greater than the number of neutrophils at normal doses, which causes excessive nanoparticles to reach nontargeted organs or tissues, leading to serious adverse effects. To address these problems, a neutrophil-targeting delivery system (DiR-DADGC-L) based on DiR-labeled and butanedioic acid (DA)-linked 5-amino-3,5-dideoxy-D-Glycerol-D-galactonanulose-cholesterol conjugate (DADGC) was designed to improve the efficiency of hitchhiking neutrophils through the specific binding of sialic acid (SA) to L-selectin (SA-binding receptor, expressed on neutrophils). DiR-DADGC-L was prepared with favorable particle size and encapsulation efficiency (%EE) to deliver DiR into neutrophils. Subsequently, diverse doses of DiR-DADGC-L were injected intravenously into S180 tumor-bearing and cyclophosphamide-depleted (CTX-D) S180 tumor-bearing mice to evaluate the in vivo behavior of liposomes. The results verified the following: a) The content of DiR-DADGC-L in neutrophils accounts for approximately 14.5% of the content of DiR-DADGC-L in plasma, and the uptake capacity of neutrophils remains unchanged under different doses, and b) both neutrophils and the enhanced permeability and retention (EPR) effect might exert significant roles in tumor treatment. As for the neutrophil-mediated delivery system, higher doses are not necessarily appropriate, and a lower dose may achieve an unexpected effect. It will be wise to determine an optimum dose to improve delivery efficiency.

Targeted delivery of zoledronic acid through the sialic acid - Siglec axis for killing and reversal of M2 phenotypic tumor-associated macrophages - A promising cancer immunotherapy.

Immune checkpoint inhibitors (ICIs), like monoclonal antibodies of PD-1, CTLA-4, and their ligands, are effective only in some populations of patients with cancer, because the immunosuppressive state of the tumor microenvironment (TME) in some patients cannot be effectively reversed after ICI therapy. Sialic acid (SA) receptors in the Siglec family are highly expressed on the surface of tumor-associated macrophages (TAMs) and most have immunosuppressive effects. Therefore, targeting TAMs (the siglec axis) to reverse tumor immunosuppression may provide a new direction for the development of novel tumor immunotherapies. We designed a Zoledronic acid (ZA)-loaded liposome modified by a SA-octadecylamine conjugate (ZA-SL) to act as a novel nanomedicine delivery platform. This platform can efficiently deliver ZA to TAMs through the combination of SA and Siglec-1 and exerts specific cytotoxicity or phenotypic remodeling of M2-like TAMs depending on the drug concentration in TAMs. In vivo experiments showed that ZA-SL had good TAM targeting ability, and after treatment, the S180 tumors of mice were significantly inhibited, and the proportion of M1-like TAMs was significantly higher than that of M2-like TAMs with no significant adverse reactions in mice. Therefore, SA-modified ZA-loaded liposomes may provide a promising strategy for cancer immunotherapy.

Endogenous oxygen generating multifunctional theranostic nanoplatform for enhanced photodynamic-photothermal therapy and multimodal imaging.

Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), has been considered as a noninvasive option for cancer therapy. However, insufficient penetration depth, tumor hypoxia, and a single treatment method severely limit the effectiveness of treatment. Methods: In this study, a multifunctional theranostic nanoplatform has been fabricated based on Au/Ag-MnO2 hollow nanospheres (AAM HNSs). The Au/Ag alloy HNSs were first synthesized by galvanic replacement reaction and then the MnO2 nanoparticles were deposited on the Au/Ag alloy HNSs by the reaction between Ag and permanganate (KMnO4), finally obtained the AAM HNSs. Then, SH-PEG was modified on the surface of AAM HNSs by the interaction of sulfhydryl and Au/Ag alloy, which improved the dispersibility and biocompatibility of the HNS. Next, the PDT photosensitizer Ce6 was loaded into AAM HNSs, benefiting from the hollow interior of the structure, and the AAM-Ce6 HNSs were obtained. Results: The AAM HNSs exhibit broad absorption at the near infrared (NIR) biological window and remarkable photothermal conversion ability in the NIR-II window. The MnO2 nanoparticles can catalyze endogenous H2O2 to generate O2 and enhance the therapeutic effect of PDT on tumor tissue. Simultaneously, MnO2 nanoparticles intelligently respond to the tumor microenvironment and degrade to release massive Mn2+ ions, which introduce magnetic resonance imaging (MRI) properties. When AAM-Ce6 HNSs are loaded with Ce6, the AAM-Ce6 HNSs can be used for triple-modal imaging (fluorescence/photoacoustic/magnetic resonance imaging, FL/PAI/MRI) guided combination tumor phototherapy (PTT/PDT). Conclusion: This multifunctional nanoplatform shows synergistic therapeutic efficacy better than any single therapy by achieving multimodal imaging guided cancer combination phototherapy, which are promising for the diagnosis and treatment of cancer.

A Sialylated-Bortezomib Prodrug Strategy Based on a Highly Expressed Selectin Target for the Treatment of Leukemia or Solid Tumors.

PURPOSE: This study aimed to explore the potential of sialic acid - related selectin targeting strategy in the treatment of leukemia and some solid tumors. We expected it could "actively" bind tumor cells and kill them, reducing non-specific toxicity to normal cells. METHODS: BOR-SA prodrug was synthesized by reacting an ortho-dihydroxy group in SA with a boronic acid group in BOR. Two kinds of leukemia cells (RAW264.7 and HL60 cells), one solid sarcoma cell model (S180 cells) and their corresponding normal cells (monocytes (MO), neutrophil (NE) and fibroblast (L929)) were selected for the in vitro cell experiments (cytotoxicity, cellular uptake, cell cycle and apoptosis experiments). The S180 tumor-bearing Kunming mice model was established for anti-tumor pharmacodynamic experiments. RESULTS: In vitro cell assay results showed that uptake of BOR-SA by HL60 and S180 cells were increased compared with the control group. BOR-SA induced a lower IC50, higher ratio of apoptosis and cell cycle arrest of tumor cells. In vivo anti-S180 tumor pharmacodynamics experiments showed that mice in the BOR-SA group had higher tumor inhibition rate, higher body weight and lower immune organ toxicity compared with the control group. CONCLUSIONS: sialic acid-mediated selectin targeting strategy may have great potential in the treatment of related tumors.

Oyster Versatile IKKα/ßs Are Involved in Toll-Like Receptor and RIG-I-Like Receptor Signaling for Innate Immune Response.

IκB kinases (IKKs) play critical roles in innate immunity through signal-induced activation of the key transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factors (IRFs). However, studies of invertebrate IKK functions remain scarce. In this study, we performed phylogenetic analysis of IKKs and IKK-related kinases encoded in the Pacific oyster genome. We then cloned and characterized the oyster IKKα/ß-2 gene. We found that oyster IKKα/ß-2, a homolog of human IKKα/IKKß, responded to challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)]. As a versatile immune molecule, IKKα/ß-2 activated the promoters of NF-κB, TNFα, and IFNß, as well as IFN-stimulated response element (ISRE)-containing promoters, initiating an antibacterial or antiviral immune state in mammalian cells. Importantly, together with the cloned oyster IKKα/ß-1, we investigated the signal transduction pathways mediated by these two IKKα/ß proteins. Our results showed that IKKα/ß-1 and IKKα/ß-2 could interact with the oyster TNF receptor-associated factor 6 (TRAF6) and that IKKα/ß-2 could also bind to the oyster myeloid differentiation factor 88 (MyD88) protein directly, suggesting that oyster IKKα/ßs participate in both RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling for the reception of upstream immune signals. The fact that IKKα/ß-1 and IKKα/ß-2 formed homodimers by interacting with themselves and heterodimers by interacting with each other, along with the fact that both oyster IKKα/ß proteins interacted with NEMO protein, indicates that oyster IKKα/ßs and the scaffold protein NEMO form an IKK complex, which may be a key step in phosphorylating IκB proteins and activating NF-κB. Moreover, we found that oyster IKKα/ßs could interact with IRF8, and this may be related to the IKK-mediated activation of ISRE promotors and their involvement in the oyster "interferon (IFN)-like" antiviral pathway. Moreover, the expression of oyster IKKα/ß-1 and IKKα/ß-2 may induce the phosphorylation of IκB proteins to activate NF-κB. These results reveal the immune function of oyster IKKα/ß-2 and establish the existence of mollusk TLR and RLR signaling mediated by IKKα/ß proteins for the first time. Our findings should be helpful in deciphering the immune mechanisms of invertebrates and understanding the development of the vertebrate innate immunity network.

Ultrasensitive photoelectrochemical biosensor for the detection of HTLV-I DNA: A cascade signal amplification strategy integrating λ-exonuclease aided target recycling with hybridization chain reaction and enzyme catalysis.

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h1DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h1DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h2DNA and h3DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels.

Conservation and divergence of mitochondrial apoptosis pathway in the Pacific oyster, Crassostrea gigas.

Apoptosis is considered a crucial part of the host defense system in oysters according to previous reports; however, the exact process by which this occurs remains unclear. Besides, mitochondrial apoptosis is the primary method of apoptosis in vertebrate cells, but has been poorly studied in invertebrates and is quite controversial. In this study, we investigated the molecular mechanism of mitochondrial apoptosis in the Pacific oyster Crassostrea gigas. Notably, we show that most key elements involved in the vertebrate mitochondrial apoptosis pathway - including mitochondrial outer membrane permeabilization, cytochrome c release, and caspase activation - are also present in C. gigas. In contrast, the lack of Bcl-2 homology 3-only subfamily members and apoptotic protease activating factor-1 (APAF-1) protein revealed evolutionary diversity from other phyla. Our results support that mitochondrial apoptosis in animals predates the emergence of vertebrates, but suggest that an unexpectedly diverse mitochondrial apoptosis pathway may exist in invertebrates. In addition, our work provided new clues for an improved understanding of how bivalve acclimate themselves to an inconstant environment.

Molecular characterization and functional analysis of tumor necrosis factor receptor-associated factor 2 in the Pacific oyster.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a family of crucial adaptors, playing vital roles in mediating signal transduction in immune signaling pathways, including RIG-I-like receptor (RLR) signaling pathway. In the present study, a new TRAF family member (CgTRAF2) was identified in the Pacific oyster, Crassostrea gigas. Comparison and phylogenetic analysis revealed that CgTRAF2 could be a new member of the invertebrate TRAF2 family. Quantitative real-time PCR revealed that CgTRAF2 mRNA was highly expressed in the digestive gland, gills, and hemocytes, and it was significantly up-regulated after Vibrio alginolyticus and ostreid herpesvirus 1 (OsHV-1) challenge. The CgTRAF2 mRNA expression profile in different developmental stages of oyster larvae suggested that CgTRAF2 could function in early larval development. CgTRAF2 mRNA expression pattern, after the silence of CgMAVS (Mitochondrial Antiviral Signaling) -like, indicated that CgTRAF2 might function downstream of CgMAVS-like. Moreover, the subcellular localization analysis revealed that CgTRAF2 was localized in cytoplasm, and it may play predominately important roles in signal transduction. Collectively, these results demonstrated that CgTRAF2 might play important roles in the innate immunity and larval development of the Pacific oyster.