Formulation-Driven Control of mRNA Polyplex Physicochemical Properties Enables Spleen-Targeted Systemic Delivery.

Impact of tail unsaturation in ionizable lipids on mRNA delivery efficiency and immunogenicity of lipid nanoparticles.

Polymers represent a promising therapeutic platform for extrahepatic messenger RNA (mRNA) delivery but are hampered by low in vivo efficacy due to polyplex serum instability and inadequate endosomal escape following systemic administration. Here, we report the rational design and combinatorial synthesis of zwitterionic phospholipidated polymers (ZPPs) via cationic polymer postmodification by alkylated dioxaphospholane oxides to deliver mRNA to spleen and lymph nodes in vivo. This modular postmodification approach readily produces tunable zwitterionic species for serum resistance and introduces alkyl chains simultaneously to enhance endosomal escape, thereby transforming deficient cationic polymers to efficacious zwitterionic mRNA carriers without the need to elaborately synthesize functional monomers. ZPPs mediated up to 39 500-fold higher protein expression than their parent cationic counterparts in vitro and enabled efficacious mRNA delivery selectively in spleen and lymph nodes following intravenous administration in vivo. This zwitterionic phospholipidation methodology provides a versatile and generalizable postmodification strategy to introduce zwitterions into the side chains of cationic polymers, extending the utility of cationic polymer families for precise mRNA delivery and demonstrating substantial potential for immunotherapeutic applications.

Described here is the synthesis and assembly of a new-generation mRNA-bifunctional lipid nanoparticle (mRNA-BLNP3) for selective delivery of mRNA to antigen-presenting cells (APCs). Compared to mRNA-BLNP1 and BLNP2 (BioRxiv,doi.org/10.1101/2023.12.26.572282), the mRNA-BLNP3, incorporating a glycolipid with the lipid moiety designed to target the CD1d receptor on dendritic cells (DCs) and the sugar head group targeting the mannose-binding receptors on APCs is more selective with stronger target-specific immune responses. It was shown that vaccination in mice with mRNA-BLNP1 elicited enhanced cytokine induction and antibody responses compared to traditional mRNA-LNPs, and the mRNA-BLNP2 vaccine, incorporating a mannose-glycolipid, further improved DC targeting. However, BLNP3, incorporating the glycolipid with an aryl-mannose head group targeting the mannose receptor on APCs and the same lipid moiety targeting the Cd1d receptor on DCs, showed superior lymph node targeting in vivo with reduced liver accumulation, enhanced mRNA expression in DCs and macrophages, and increased DC maturation. Immunization in mice with mRNA-BLNP3 elicited enhanced humoral and cellular immune responses compared to mRNA-BLNP1 and mRNA-BLNP2, with higher antigen-specific IgG titers and granzyme B–producing CD8+ T cells, demonstrating that BLNP3 is a promising bifunctional lipid nanoparticle for delivery of mRNA vaccines with improved efficacy and safety.

Modified polyethylenimines as non-viral gene delivery systems for aerosol gene therapy: investigations of the complex structure and stability during air-jet and ultrasonic nebulization

A reduced capacity of antigen presenting cells (APC) to provide pro-T helper 1 (Th1) signals, such as IL-12, to T cells during early life may be implicated in the development of T helper 2 (Th2)-mediated allergic disease. In this study we examined the relationships between the capacity for IL-12 responses in the neonatal period and atopic risk (family allergy), in vitro T cell responses to allergens, and the subsequent development of allergic disease at 6 years. The capacity of circulating neonatal (and maternal) APC to produce IL-12 p70 in response to LPS (and IFN-gamma) stimulation was assessed in a group of 60 children with previously well-characterized immune responses to allergens and atopic outcomes. The IL-12 responses were compared with allergen-induced lymphoproliferation (to house dust mite (HDM) ovalbumin (OVA), cat and beta-lactoglobulin (BLG)) and IL-13 and IFN-gamma cytokine responses (to OVA, HDM and phytohaemaglutinin (PHA)) in the neonatal and postnatal periods. IL-12 responses were also compared according to atopic risk and atopic outcomes (doctor-diagnosed asthma, eczema, food allergies and sensitization as evidenced by skin prick testing) at 6 years clinical follow-up. Maternal peripheral blood mononuclear cells (PBMC) synthesized significantly greater amounts of IL-12 than neonatal PBMC, though within maternal-infant pairs IL-12 responses were significantly correlated (r = 0.4, P = 0.019). Moreover, neonatal IL-12 responses were positively correlated with neonatal allergen proliferation for HDM (r = 0.6, P < 0.0001), OVA (r = 0.55, P < 0.0001), cat (r = 0.5, P = 0.003) and BLG (r = 0.55, P = 0.001), but negatively correlated with neonatal IL-13 responses to both allergens tested (HDM: r = - 0.4, P = 0.03 and OVA: r = - 0.5, P = 0.001). Both neonatal and maternal IL-12 responses were positively correlated with postnatal IFN-gamma responses to HDM at 12, 18 and 24 months of age (responses after age of 2 years were not assessed). There was no relationship between atopic risk and IL-12 capacity in the neonatal period, but there was a (non-significant) trend for neonatal IL-12 responses to be lower in the high-risk children who developed clinical allergy at 6 years (compared with the low risk group) although the number in this analysis was small. Reduced APC IL-12 production in the perinatal period was associated with reduced T cell activation (lymphoproliferation), stronger neonatal Th2 responses, and weaker Th1 responses to allergen in the postnatal period. This supports the notion that variations in APC function in early life may contribute to altered allergen-specific cytokine responses associated with later allergy.

In recent years, the application of mRNA vaccine-based tumor immunotherapy invigorated anti-tumor therapy. However, the low efficiency of mRNA delivery and the lack of targeting ability in vivo are the major obstacles to achieving highly efficient immunotherapy. In this work, we report a chemical library of amphiphilic carbon dots (ACDs) and the synthesized ACDs were applied to mRNA delivery, bio-imaging, and tumor immunotherapy. The ACDs can smoothly bind with mRNA to form ACDs@mRNA nanocomplexes, and the fluorescent properties of the ACDs afforded the nanoparticles with bio-imaging ability. By screening of the ACDs, O12-Tta-CDs were found to have optimal mRNA transfection efficiency and the ability of spleen-targeted delivery. In addition, O12-Tta-CDs can well transfect the immune cells and promote the maturation and antigen presentation of bone marrow-derived dendritic cells (BMDCs). Furthermore, O12-Tta-CDs@OVA-mRNA was successfully applied to inhibit tumor growth, and more specific T-cell infiltration was observed in spleen and tumors of mice after treatment in the E.G7-OVA tumor model. Besides, O12-Tta-CDs@OVA-mRNA also achieved a good therapeutic effect in tumor recurrence inhibition and tumor prophylactic experiments. This study provided a new direction for the design of mRNA vectors, which is promising in tumor immunotherapy.

Asthma is an inflammatory disease that affects many people around the world, especially persons at paediatric age group. The effectiveness of tyrosol, a natural phenolic compound, was examined in the asthma model induced by ovalbumin (OVA). For this purpose, four groups, each consisting of eight rats, were arranged. For 21days, physiological saline solution was treated to the control group and OVA was treated to the groups of OVA, OVA + dexamethasone (Dexa) and OVA + tyrosol groups, intraperitoneally and through inhalation. Additionally, 0.25mg/kg Dexa was treated to the OVA + Dexa group and 20mg/kg tyrosol to the OVA + tyrosol group by oral gavage. Serum, blood, bronchoalveolar lavage fluid (BALF) and lung tissues of the rats were examined. It was observed that MDA level decreased, GSH level and GPx activity increased, and there was no change in CAT activity in lung tissues of the tyrosol treatment groups. It was also observed that NF-κB, TNF-α, IL-4, IL-5, IL-13, IFN-γ and IgE levels decreased compared to the OVA group in lung tissue and serum samples except for serum NF-κB and IL-4. However, no effect on IL-1 β level was observed. In addition, it was determined that tyrosol treatment increased the IL-10 level on both tissue samples. The results of the histopathological investigation of lung tissue showed that tyrosol significantly ameliorated OVA-induced histopathological lesions. Additionally, PAS staining showed that mucus hypersecretion was significantly reduced with the use of tyrosol. In addition, it was determined that the number of eosinophils decreased significantly in blood and BALF samples. The obtained results showed that tyrosol possessed antioxidant and anti-inflammatory features on OVA-induced rats and preserved tissue architecture.

Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4+ T cells in the regulation of T cell differentiation

Although anti−cancer immuno−based combinatorial therapeutic approaches have shown promising results, efficient tumour eradication demands further intensification of anti−tumour immune response. With the emerging field of nanovaccinology, multi−walled carbon nanotubes (MWNTs) have manifested prominent potentials as tumour antigen nanocarriers. Nevertheless, the utilization of MWNTs in co−delivering antigen along with different types of immunoadjuvants to antigen presenting cells (APCs) has not been investigated yet. We hypothesized that harnessing MWNT for concurrent delivery of cytosine−phosphate−guanine oligodeoxynucleotide (CpG) and anti-CD40 Ig (αCD40), as immunoadjuvants, along with the model antigen ovalbumin (OVA) could potentiate immune response induced against OVA−expressing tumour cells. We initially investigated the effective method to co−deliver OVA and CpG using MWNT to the APC. Covalent conjugation of OVA and CpG prior to loading onto MWNTs markedly augmented the CpG−mediated adjuvanticity, as demonstrated by the significantly increased OVA−specific T cell responses in vitro and in C57BL/6 mice. αCD40 was then included as a second immunoadjuvant to further intensify the immune response. Immune response elicited in vitro and in vivo by OVA, CpG and αCD40 was significantly potentiated by their co−incorporation onto the MWNTs. Furthermore, MWNT remarkably improved the ability of co−loaded OVA, CpG and αCD40 in inhibiting the growth of OVA−expressing B16F10 melanoma cells in subcutaneous or lung pseudo−metastatic tumour models. Therefore, this study suggests that the utilization of MWNTs for the co−delivery of tumour−derived antigen, CpG and αCD40 could be a competent approach for efficient tumours eradication.

Murine Bone Marrow-Derived Megakaryocytes Are Capable of Antigen Cross-Presentation on Major Histocompatibility Class (MHC) I Molecules

BackgroundNano-sized drug delivery system has been widely studied as a potential technique to promote tumor-specific delivery of anticancer drugs due to its passive targeting property, but resulting in very restricted improvements in its systemic administration so far. There is a requirement for a different approach that dramatically increases the targeting efficiency of therapeutic agents at targeted tumor tissues.MethodsTo improve the tumor-specific accumulation of anticancer drugs and minimize their undesirable toxicity to normal tissues, a tumor-implantable micro-syringe chip (MSC) with a drug reservoir is fabricated. As a clinically established delivery system, six liposome nanoparticles (LNPs) with different compositions and surface chemistry are prepared and their physicochemical properties and cellular uptake are examined in vitro. Subsequently, MSC-guided intratumoral administration is studied to identify the most appropriate for the higher tumor targeting efficacy with a uniform intratumoral distribution. For efficient cancer treatment, pro-apoptotic anticancer prodrugs (SMAC-P-FRRG-DOX) are encapsulated to the optimal LNPs (SMAC-P-FRRG-DOX encapsulating LNPs; ApoLNPs), then the ApoLNPs are loaded into the 1 μL-volume drug reservoir of MSC to be delivered intratumorally for 9 h. The tumor accumulation and therapeutic effect of ApoLNPs administered via MSC guidance are evaluated and compared to those of intravenous and intratumoral administration of ApoLNP in 4T1 tumor-bearing mice.ResultsMSC is precisely fabricated to have a 0.5 × 4.5 mm needle and 1 μL-volume drug reservoir to achieve the uniform intratumoral distribution of LNPs in targeted tumor tissues. Six liposome nanoparticles with different compositions of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (PS), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)2000] (PEG2000-DSPE) are prepared with average sizes of 100–120 nm and loaded into the 1 μL-volume drug reservoir in MSC. Importantly negatively charged 10 mol% of PS-containing LNPs are very slowly infused into the tumor tissue through the micro-syringe of the MSC over 6 h. The intratumoral targeting efficiency of MSC guidance is 93.5%, effectively assisting the homogeneous diffusion of LNPs throughout the tumor tissue at 3.8- and 2.7-fold higher concentrations compared to the intravenous and intratumoral administrations of LNPs, respectively. Among the six LNP candidates 10 mol% of PS-containing LNPs are finally selected for preparing pro-apoptotic SMAC-P-FRRG-DOX anticancer prodrug-encapsulated LNPs (ApoLNPs) due to their moderate endocytosis rate high tumor accumulation and homogenous intratumoral distribution. The ApoLNPs show a high therapeutic effect specifically to cathepsin B-overexpressing cancer cells with 6.6 μM of IC50 value while its IC50 against normal cells is 230.7 μM. The MSC-guided administration of ApoLNPs efficiently inhibits tumor growth wherein the size of the tumor is 4.7- and 2.2-fold smaller than those treated with saline and intratumoral ApoLNP without MSC, respectively. Moreover, the ApoLNPs remarkably reduce the inhibitor of apoptosis proteins (IAPs) level in tumor tissues confirming their efficacy even in cancers with high drug resistance.ConclusionThe MSC-guided administration of LNPs greatly enhances the therapeutic efficiency of anticancer drugs via the slow diffusion mechanism through micro-syringe to tumor tissues for 6 h, whereas they bypass most hurdles of systemic delivery including hepatic metabolism, rapid renal clearance, and interaction with blood components or other normal tissues, resulting in the minimum toxicity to normal tissues. The negatively charged ApoLNPs with cancer cell-specific pro-apoptotic prodrug (SMAC-P-FRRG-DOX) show the highest tumor-targeting efficacy when they are treated with the MSC guidance, compared to their intravenous or intratumoral administration in 4T1 tumor-bearing mice. The MSC-guided administration of anticancer drug-encapsulated LNPs is expected to be a potent platform system that facilitates overcoming the limitations of systemic drug administration with low delivery efficiency and serious side effects.Graphical

BackgroundThere have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.MethodsIn FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c+ MHC class II+ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c+ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).ResultsIn WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c+ MHC class II+ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c+ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.ConclusionAntigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.

수지상세포는 종양면역에서 필수적인 강력한 CTL 반응을 개시할 수 있는 유일한 세포이다 . 특히 외인성 종양항원에 대한 CTL 반응 유도는 활성화된 수지상세포의 IL-12 분비를 통한 CD4+ helper T세포의 cross-priming을 필요로 한다. 그러나 최근에 활성화된 수지상세포는 <TEX>$Th_1$</TEX> 면역반응을 유도하지만 활성화 시간이 경과함에 따라 오히려 <TEX>$Th_2$</TEX>반응을 유도 할 수 있다. 따라서 본 연구에서는 OVA를 종양항원 모델로 설정하여 종양특이적인 CTL 반응을 형성하기 위한 최적의 수지상세포 활성화 조건을 조사하였다. 마우스 골수세포에 서 수지상세포로의 분화는 항원제시 기능을 위한 표면분자의 발현 측면에서 볼 때 배양 6일-7일 정도가 적합하였다. 수지상세포의 IL-12 생성능은 배양 6일 이상, OVA 항원 탑재 8시간 이상의 경우에 연이은 LPS 성숙자극으로 오히려 감소하는 경향을 보였다. 즉 배양 6일의 수지상세포에 OVA 항원 탑재를 8시간 수행한 경우(8-h DC)가 in vitro에서의 IL-12생성능, ex vivo에서의 세포내 <TEX>$IFN-{\gamma}$</TEX>를 발현하는 CD8+ T세포의 증가 및 OVA 특이적인 세포독성효과 등에서 가장 좋은 결과를 보였다. 또한 in vivo에서 종양 치료 및 예방효과에서도 8-h DC로 면역한 경우에 가장 우수한 종양형성 억제 효과와 생존기간 연장효과를 보였다. 현재 대부분의 수지상 세포를 이용한 항종양 백신에서 항원 탑재반응을 24시간 동안 수행하고 있으나, 본 실험의 결과로 볼 때, 8시간의 in vitro 항원 탑재가 보다 효과적인 종양특이적 CTL 반응과 항종양 면역반응을 유도함을 알 수 있다. 결론적으로 본 연구를 통하여 8시간 이상의 항원접촉은 수지상세포의 기능적 활성능력을 오히려 고갈시킬 수 있음을 제시한다. Dendritic cells (DCs) are the only antigen presenting cells (APCs) capable of initiating immune responses, which is crucial for priming the specific cytotoxic T lymphocyte (CTL) response and tumor immunity. Upon activation by DCs, CD4+ helper T cells can cross-prime CD8+ CTLs via IL-12. However, recently activated DCs were described to prime in vitro strong T helper cell type 1 <TEX>$(Th_1)$</TEX> responses, whereas at later time points, they preferentially prime <TEX>$Th_2$</TEX> cells. Therfore, we examined in this study the optimum kinetic state of DCs activation impacted on in vivo priming of tumor-specific CTLs by using ovalbumin (OVA) tumor antigen model. Bone-marrow-derived DCs showed an appropriate expression of surface MHC and costimulatory molecules after 6 or 7-day differentiation. The 6-day differentiated DCs pulsed with OVA antigen for 8 h (8-h DC) and followed by restimulation with LPS for 24 h maintained high interleukin (IL)-12 production potential, accompanying the decreased level in their secretion by delayed re-exposure time to LPS. Furthermore, immunization with 8-h DC induced higher intracellular <TEX>$interferon(IFN)-{\gamma}+/CD8+T$</TEX> cells and elicited more powerful cytotoxicity of splenocytes to EG7 cells, a clone of EL4 cells transfected with an OVA cDNA, than immunization with 24-h DC. In the animal study for the evaluation of therapeutic or protective antitumor immunity, immunization with 8-h DC induced an effective antitumor immunity against tumor of EG7 cells and completely protected mice from tumor formation and prolonged survival, respectively. The most commonly used and clinically applied DC-based vaccine is based on in vitro antigen loading for 24 h. However, our data indicated that antigen stimulation over 8 h decreased antitumor immunity with functional exhaustion of DCs, and that the 8-h DC would be an optimum activation state impacted on in vivo priming of tumor-specific CTLs and subsequently lead to induction of strong antitumor immunity.

Efficient capture and presentation of tumor antigens by antigen-presenting cells (APCs), especially dendritic cells (DCs), are crucial for activating the anti-tumor immunity. However, APCs are immunosuppressed in the tumor microenvironment, which hinders the tumor elimination. To reprogram APCs for inducing strong anti-tumor immunity, we report here a co-delivery immunotherapeutic strategy targeting the phagocytosis checkpoint (signal regulatory protein α, SIRPα) and stimulator of interferon genes (STING) of APCs to jointly enhance their ability of capturing and presenting tumor antigens. In brief, a small interfering RNA targeting SIRPα (siSIRPα) and a STING agonist (cGAMP) were co-delivered into APCs by the encapsulation into poly(ethylene glycol)-b-poly(lactide-co-glycolide)-based polymeric nanoparticles (NPsiSIRPα/cGAMP). siSIRPα-mediated SIRPα silence promoted APCs to actively capture tumor antigens by engulfing tumor cells. The cGAMP-stimulated STING signaling pathway further enhanced the functions of APCs, thereby increased the activation and expansion of CD8+ T cells. Using ovalbumin (OVA)-expressing melanoma as a model, we demonstrated that NPsiSIRPα/cGAMP stimulated the activation of OVA-specific CD8+ T cells and induced holistic anti-tumor immune responses by reversing the immunosuppressive phenotype of APCs. Collectively, this co-delivery strategy synergistically enhanced the functions of APCs and can be extended to the treatment of tumors with poor immunogenicity.

BackgroundDevelopment of a potent vaccine adjuvant without introduction of any side effects remains an unmet challenge in the field of the vaccine research.Methodology/Principal FindingsWe found that laser at a specific setting increased the motility of antigen presenting cells (APCs) and immune responses, with few local or systemic side effects. This laser vaccine adjuvant (LVA) effect was induced by brief illumination of a small area of the skin or muscle with a nondestructive, 532 nm green laser prior to intradermal (i.d.) or intramuscular (i.m.) administration of vaccines at the site of laser illumination. The pre-illumination accelerated the motility of APCs as shown by intravital confocal microscopy, leading to sufficient antigen (Ag)-uptake at the site of vaccine injection and transportation of the Ag-captured APCs to the draining lymph nodes. As a result, the number of Ag+ dendritic cells (DCs) in draining lymph nodes was significantly higher in both the 1° and 2° draining lymph nodes in the presence than in the absence of LVA. Laser-mediated increases in the motility and lymphatic transportation of APCs augmented significantly humoral immune responses directed against a model vaccine ovalbumin (OVA) or influenza vaccine i.d. injected in both primary and booster vaccinations as compared to the vaccine itself. Strikingly, when the laser was delivered by a hair-like diffusing optical fiber into muscle, laser illumination greatly boosted not only humoral but also cell-mediated immune responses provoked by i.m. immunization with OVA relative to OVA alone.Conclusion/SignificanceThe results demonstrate the ability of this safe LVA to augment both humoral and cell-mediated immune responses. In comparison with all current vaccine adjuvants that are either chemical compounds or biological agents, LVA is novel in both its form and mechanism; it is risk-free and has distinct advantages over traditional vaccine adjuvants.