Research paperIntranasal delivery of cationic liposome-protamine complex mRNA vaccine elicits effective anti-tumor immunity
Graphical abstract
Introduction
Malignant tumors are associated with the highest morbidity and mortality rates among diseases [1]. Tumor immunotherapy refers to immunization with tumor-related antigens to stimulate an immune response to inhibit the proliferation and spread of tumor cells in order to prevent tumor growth [2]. However, the safety and effectiveness of tumor immunotherapies are less than optimal [3]. With the continued development of tumor immunotherapies, autologous tumor vaccines have attracted widespread attention as an emerging strategy with potential anti-cancer therapeutic effects. Protein, peptide and DNA coding plasmids are used as conventional antigens for vaccine development [4]. As compared to conventional vaccination methods, mRNAs encoding tumor-associated antigens can be administered as mRNA-based anti-cancer vaccines [5], [6]. Peptide and protein vaccines are comparatively easy to prepare for large-scale production, but the immunogenicity is weak, the duration of the immune response is short, and the selection of antigen peptides is limited [7], [8]. Unlike DNA, mRNA can gain entry into the cytoplasm and does not run the risk of uncontrolled gene integration [9]. Nonetheless, it is difficult for a negatively charged mRNA molecule to enter the cell through the negatively charged cell membrane. In addition, naked mRNA molecules are easily degraded by plasma or tissue enzymes. To overcome these problems, mRNA-based vaccines can be administered by injection of mRNA-loaded dendritic cells (DCs) [10]. However, this strategy is costly and the process cannot mass produce therapeutic vaccines. Therefore, the construction of a suitable mRNA tumor vaccine delivery system, improvement of mRNA stability, promotion of cellular antigen presentation, and the induction of an anti-tumor immune response have become particularly important strategies to enhance the effect of mRNA-based anti-tumor vaccines. A possible strategy to overcome this problem is to encapsulate the mRNA in nanoparticles. In fact, the delivery of mRNA nanoparticles has attracted the interest of many research groups in recent years.
Cationic liposomes are effective adjuvants for vaccine delivery and can enhance the immunogenicity of various antigens to elicit both cellular immune and humoral responses [11]. The adjuvant effect of cationic liposomes has been attributed to the selective uptake of cationic liposome antigens by regional lymph nodes for efficient antigen aggregation and presentation to achieve massive delivery to antigen-presenting cells (APC) [12], [13]. Liposomes are composed of phospholipid bilayers that allow to encapsulate various antigens or immunostimulatory agents [14]. DOTAP can directly contact the cell surface through electrostatic action, is easily taken up by DCs, induces DCs to present antigens, can enhance cellular immune response, and cholesterol added to DOTAP lipid can have a stable effect on liposomes, Polyethylene glycol (PEG) can limit opsonization and non-specific uptake, increase circulation half-life in the body[5], improve the passage of nanoparticles through mucus, and induce a stronger mucosal response[15]. However, during the process of cationic liposome-encapsulated antigen production, the encapsulated mRNA antigen can be easily lost and the stability of mRNA is compromised by the presence of nucleases both in vitro and in vivo. Protamine is a positively charged polycation that can be used to condense mRNA into nano-sized complexes to prevent nuclease degradation [16], [17]. The purpose of adding protamine is to be able to compress mRNA and be completely encapsulated by liposomes. The main advantage of the cationic LPC, as compared to single liposome or protamine complexes, is that the former can compress and completely encapsulate mRNA by liposomes, rendering the formulation more stable, showing that the accumulation of antigen in APC in the spleen and lymph nodes is a clear advantage. We hypothesize that the cationic LPC would act as an effective mRNA vaccine delivery system.
The immune pathway is key to activate an immune response in response to vaccines. Current mRNA vaccines are injected intradermally, intravenously, or subcutaneously. However, these routes limit the migration of nanoparticles. More recently, the mucosal route has been proposed as a viable strategy for the delivery of molecules to initiate an immune reaction. As compared with traditional administration methods, the intranasal route not only effectively induces a systemic immune response, but also local mucosal immunity [18]. Intranasal administration can reach nasal-associated lymphoid tissue (NALT), which contains specialized M-like cells, DCs, and large numbers of B and T lymphocytes [19]. M-like cell refers to the follicle-associated epithelium cells, which is a special kind of epithelial cells, M cells can present antigens to DCs through transcellular action, and have the function of actively recognizing antigens through phagocytosis and transporting them to APC, eventually initiating systemic and mucosal responses [11], [19], [20]. Therefore, NALT is an appropriate site for antigen internalization to induce protective immunity against cancer cells [21]. However, the capacity of nasal mucosal epithelial cells to take up antigens is weak, which results in a poor immune response [22]. In addition, nasal mucosal epithelial cells cannot maintain a sustained long-acting effect. To address this problem, a recent study reported that use of microspheres for intranasal delivery can significantly improve the vaccine-induced immune response [23]. Related studies have shown that nanoparticle-encapsulated mRNA-encoded ovalbumin (OVA) promotes a specific immune response via cytotoxic T lymphocytes (CTLs) against E.G7-OVA tumor cells, indicating that tumor immunity can be achieved by nasal administration of mRNA [24].
Based on the above rationale, we speculated that cationic LPC may be appropriate as an mRNA-based nasal delivery system to induce a potent immune response. Therefore, we set up an immunization scheme with the use of cytokeratin 19 (CK19) as an antigen. CK19 is a differentiation-specific protein that is widely distributed on epithelial cell membranes and is prevalent in lung cancer cells. Hence, the CK19 protein can be used as a target antigen for immunotherapy against lung cancer [25], [26], [27]. Our group first designed a cationic LPC as a safe and effective intranasal mRNA-encoded CK19 (mCK19) vaccine delivery system to elicit an effective anti-tumor immune response. Here, we systematically evaluated the composition and morphology of the cationic liposome/protamine mRNA vaccine. Cationic liposome/protamine-encapsulated mRNA vaccines were compared with protamine or cationic liposome groups to assess the uptake by and activation of DCs to elicit a cellular immune response. The anti-tumor immunity therapeutic immunotherapy models were evaluated with the use of Lewis lung cancer cells (LLC).
Section snippets
Materials, cell lines
The cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethy leneglycol)-2000 (DSPE-PEG-2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Protamine sulfate (grade X) and cholesterol were obtained from Sigma-Aldrich Co. (Gillingham, Dorset, UK). A CCK-8 cell proliferation kit was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Fetal bovine serum (FBS), penicillin, and
Preparation and characterization of LPC encapsulated mRNA
To construct functional mRNA transcripts, a DNA vector (pGP-T7-MCS-polyA) containing the CK19 gene was constructed, as illustrated in Fig. 2a. Purified CK19 mRNA electrophoresis results showed that the transcribed CK19 was about 1381 nucleotides in length Fig. 2b (the 2b (a) is the band of CK19 in vitro transcription, the 2b (b) is a DNA marker), which was consistent with the predicted length, thereby demonstrating successful CK19 transcription. Next, the capacity of LPC/mRNA to overcome the
Conclusion
In this study, a cationic LPC was constructed to deliver in vitro transcribed mRNA for cancer immunotherapy. LPC/mRNA effectively promoted antigen uptake by DCs, effectively stimulated DC maturation, and promoted cytokine secretion, resulting in a cellular immune response. More importantly, LPC/mCK19 exhibited promising tumor therapy effects in an aggressive LLC model. Therefore, LPC/mCK19 is a promising vaccine delivery candidate for immunotherapy through the nasal mucosa to produce effective
Acknowledgements
This study was partially funded by the National Natural Science Foundation (grant no. NSFC81460541).
Compliance with ethical standards
Conflict of interest: All authors declare that they have no conflicts of interest in this work.
Ethical approval: All experiments involving animals were conducted in accordance with the Chinese version of the Guide for the Care and Use of Laboratory Animals and the guidelines of the Experimental Animal Center of Ningxia Medical University (approval number IACUC-NYLAC-20).
Animal source: Specific-pathogen-free C57BL/6 female mice were obtained from the Experimental Animal Center of Ningxia Medical
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