In the case of HEL, almost all HEL was detected in the FT (right graph).
Vaccine Dynamics Project, BIKEN Center for Innovative Vaccine Research and Development, The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, Japan. Mice (n = 3 per group) were anesthetized with 3.0% isoflurane. 265). In the experiments mentioned above, DOTAP-Nano was prepared in 5% glucose solution, and DOTAP-Lipo was immersed in MES-buffered saline (pH6.2) by the manufacturer. Data curation, The protein concentration in the FT solution was measured.
Cationic lipids (such as 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) have been used as antigen carriers for cancer vaccines and showed effective adjuvant activity by themselves [1821]. These two points are generally important in understanding the mechanism of action of particle adjuvants. commercially: Lipofectin (a 1:1 mixture of DOTMA [N-[1(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride] and DOPE [dioleoyl phosphatidylethanolamine]), Transfectam, and DOTAP [1,2-dioleoyloxy-3-trimethylammonium C2395 (t^c^g^t^c^g^t^t^t^t^c^g^g^c^g^c^g^c^g^c^c^g) and P21889 (t^c^g^t^c^g^a^c^g^a^t^c^g^g^c^g^c^g^c^g^c^c^g) were synthesized by GeneDesign (Osaka, Japan); ^ indicates phosphorotihoate bonds. DOTAP-Nano induced stronger T-cell responses than DOTAP-film in both CD8+ T-cell (class I) and CD4+ T-cell (class II) responses (Fig 2A). The overnight cultured supernatant was collected and subjected to mouse IFN-gamma ELISA (Mouse IFN- DuoSet ELISA kits [R&D Systems]). Copyright 2022 by Cold Spring Harbor Laboratory Press. The mixtures were ultrafiltrated to separate lipid binding DNA and free DNA in the flow through (FT). PMID: 26112463. https://doi.org/10.1371/journal.pone.0227891.g002. Validation, 
PLOS ONE promises fair, rigorous peer review, Competing interests: Some of the authors have filed a patent application (Application Number 2018-227061) related to the content of this manuscript. In most of the experiments, three mice per group (total 129 mice for immunization experiments presented in this study) were immunized with antigen (OVA, HEL: 10 g) only or with antigen plus adjuvants (CpG ODNs: 10 g, DOTAP-Nano/-Lipo: 100 g). DOTAP is one of the most widely used cationic lipids for gene transfection applications. Investigation, C57BL/6J mice were immunized with OVA (10 g), OVA plus DOTAP (100 g), and OVA/DOTAP plus the indicated CpG adjuvant (10 g) at the tail base. After seven days, splenocytes were collected and stimulated with OVA257-264 peptide and OVA protein. Click through the PLOS taxonomy to find articles in your field. Contact us, Production Facility | 700 Industrial Park Drive Alabaster, Alabama 35007-9105, Copyright 2022 Croda International Plc. Kim BK, Hwang GB, Seu YB, Choi JS, Jin KS, Doh KO. propane]:DOPE. In this case, strong immunostimulatory cytokine induction or inflammatory responses could compensate for the lack of lipidantigen interaction and result in the induction of a T-cell response, which has been shown in other originally antigen-noninteracting adjuvants such as advax [37], AS03 [38], and MF59 [39]. It has also been shown that the combination of DOTAP and CpG ODN enhanced CpG-mediated biological activities in vivo [26, 27]. (E) C57BL/6J mice were immunized at the tail base with OVA mixed with either DOTAP-Nano or DOTAP-Lipo under the indicated buffer conditions. Writing review & editing, Roles Yes Human PBMCs or mouse BM cells were plated on 96-well plates at 1 106 cells/well/200 L. Methodology, Delivery systems feature various particles consisting of synthetic polymers, liposomes, and oil emulsions [11, 12].
On the contrary, DOTAP-Lipo itself showed a size of approximately 130 nm, and the addition of OVA did not cause any changes in size, thus indicating that DOTAP-Lipo did not interact with OVA antigen (Fig 1B; right). Both DOTAP-Nano and DOTAP-Lipo worked as efficient systems for delivering D35 into mouse and human cells, thus resulting in increased IFN- production (Fig 4C). The marrow was flushed out with RPMI 1640 by using a syringe with a 25G needle. DOTAP-Nano and HEL did not show any interactions. Validation, A mixture of DOTAP-nano/antigen and DOTAP-nano/antigen/CpG were prepared by simply adding each components in 5% glucose solution and vortexed.
However, among them, the addition of D35 or C2395 induced relatively stronger CD4+ T-cell responses (Fig 3; left). https://doi.org/10.1371/journal.pone.0227891.s001. The value of Polydispersity index (PdI) indicates the mean SD of three times measurements. 2013 Oct 9;168(4):3659-64. doi: 10.1016/j.ijcard.2013.05.092. OVA-specific total IgG, IgG1, and IgG2c were clearly detected in a manner that is dependent on both antigen and adjuvant doses (Fig 6C). However, we could not rule out the possibility of the weak interaction between DOTAP-Nano and OVA in MES-buffered conditions (potentially reflected in the slight increase of size shown in Fig 1D) also influencing the adjuvanticity of DOTAP-Nano for the induction of CD4+ T-cell responses in MES buffer (Fig 1E).
Writing original draft, Representative results are shown in S2 Fig. The DLS measurement of the interaction between lipid and antigen also confirmed that HEL antigen did not interact with DOTAP-Nano, whereas OVA antigen interacted with DOTAP-Nano in Glu/PBS (Fig 5B). To confirm that these lipids and nucleic acid interactions are functional, mouse BM cells or human PBMCs were stimulated with D35 only or in combination with D35 plus either DOTAP-Nano or DOTAP-Lipo. However, all these reagents are costly; it can be less expensive to purchase purified lipids and formulate Red blood cells were lysed with ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA), and cells were washed with RPMI 1640 and suspended in R-10 medium (RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/mL penicillin, and 100 g/mL streptomycin). Female C57BL/6 or BALB/c mice aged 410 weeks old were used for all experiments. Investigation, The abovementioned results showed that buffer conditions influenced the interaction between DOTAP and antigen and the resultant induction of T-cell responses. Vaccine Dynamics Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, Roles These results suggested that microfluidic-prepared DOTAP nanoparticles plus D35 are a promising adjuvant for a vaccine that induces therapeutic T-cell responses for treating cancer and infectious diseases. 2011 Cold Spring Harbor Laboratory Press, Alert me when Updates/Comments are published, Preparation of Macromolecules and Introduction into Cells. For the induction of antigen-specific T-cell responses by vaccination, an appropriate immune adjuvant is required. e0227891. We also confirmed this result by using different antigens of HEL, which did not interact with DOTAP (Fig 5B and 5C), and the HEL DOTAP adjuvant combination did not result in the induction of T-cell responses (Fig 5A). The preparation of all lipid nanoparticles was performed at room temperature. (C) The zeta potential of lipid particles in the indicated buffer conditions was measured by a zetasizer. Interestingly, most of these are composed of lipid plus immune stimulator. 2004;21(4):257-317. doi: 10.1615/critrevtherdrugcarriersyst.v21.i4.10. After washing, goat antimouse total IgG-, IgG1-, or IgG2c-conjugated HRP (Southern Biotech) was added and incubated for 2 h at room temperature. The experiment was performed in accordance with the Regulations on Animal Experiments at Osaka University (Permit No. However, this does not alter our adherence to PLOS ONE policies on sharing data and materials. well of a six-well dish. Consistent with these interactions, both DOTAP preparations in Glu/PBS showed robust CD8+ T-cell responses (Fig 1E; left) but not in MES-buffered saline (Fig 1E; left). A representative size distribution and polydispersity index of DOTAP-Nano, DOTAP-Lipo, and DOTAP-film with or without antigen and D35 were shown in S1 Fig. These results suggested that the low adjuvanticity of DOTAP-Lipo for the induction of CD8+ T-cell responses was mainly derived from the lack of interaction with OVA antigen, which was likely caused by MES-buffer-dependent zeta potential reduction (Fig 1C and 1D). For customers located outside the United States, you will be redirected to www.sigmaaldrich.com/avanti prior to completing your purchase. Epub 2015 Jun 23. Either DOTAP-Nano or DOTAP-film was mixed with OVA antigen and was immunized under Glu/PBS buffer conditions into mice. (800) 227-0651 The DLS evaluation of the interaction between lipid and D35 showed that D35 similarly interacted with DOTAP-Nano and DOTAP-Lipo (Fig 4B). An increase in particle size indicates that the lipid particle interacts with OVA protein. In this study, we examined microfluidic-prepared DOTAP for the induction of T-cell responses against model protein antigens and compared the adjuvanticity among different preparations of DOTAP in mice. Visualization, (D) The sizes of lipid particles and lipids mixed with OVA were measured by DLS under the indicated buffer conditions. For the induction of CD4+ T-cell responses, NanoAssemblr-formulated DOTAP-Nano showed better adjuvanticity irrespective of the buffer conditions, thus suggesting that the adjuvanticity for the induction of CD4+ T-cell responses was not necessarily dependent on the interaction between adjuvant and antigen; instead, it may be dominantly dependent on the size of DOTAP particles. The size distribution was measured by dynamic light scattering (DLS), and the zeta potential was measured by particle electrophoresis with Zetasizer Nano-ZS90 (Malvern Panalytical Ltd., UK). Typical immune potentiators are pathogen-associated molecular patterns, which are mainly various toll-like receptor (TLR) agonists including poly I:C (TLR3), MPL (TLR4), and CpG (TLR9) [810]. The sizes of DOTAP-Nano only, and mixed with indicated CpG were measured under Glu/PBS buffer conditions. After washing the cells once in RPMI 1640 and counting them, BM cells were suspended in R-10 medium. Another important factor is the interaction between DOTAP nanoparticles and antigens. The levels of OVA-specific antibodies in plasma were determined by ELISA. 2550 Acton Rd
(B) The sizes of OVA only, lipid particle only, and lipid mixed with OVA were determined by the dynamic light scattering (DLS) method. https://doi.org/10.1371/journal.pone.0227891.g001. DOTAP-nano with B, C, P type CpG interaction was also evaluated by DLS and ultrafiltration. We also examined the interaction between DOTAP and OVA antigen in Glu/PBS or MES-buffered saline. Yes Immune adjuvants can be divided into two functional categories: immune potentiator and delivery system [6, 7]. The cationic lipid DOTAP has also been used as a carrier of immune potentiators, such as CpG immunostimulatory oligodeoxynucleotide (ODN), to the endosomal compartment where the CpG receptor TLR9 resides [24, 25]. DOTAP-Nano prepared with NanoAssemblr induced robust CD8+ T-cell responses (Fig 1A; left) and CD4+ T-cell responses (Fig 1A; right), whereas DOTAP-Lipo showed almost no adjuvant effect (Fig 1A). Privacy Policy | Reset Location Preferences This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The recently developed microfluidic method enables smaller (under 100 nm) formulations of lipid nanoparticles to be created [2832]; this was not achievable using the conventional lipid film hydration method [33]. The sizes of DOTAP-particle only, and mixed with antigen protein or D35 were measured under indicated buffer conditions. 
However, in the case of DOTAP-Nano plus D35, the T-cell response agonist HEL was also dependent on DOTAPantigen interaction, thus suggesting that antigen interaction is almost obligatory in the case of DOTAP nanoparticle adjuvant. In the case of P21889, the addition of this CpG ODN was even suppressive, particularly for the induction of CD8+ T-cell responses (Fig 3; right). One is referred to as DOTAP-Nano, which was prepared by microfluidic methods using the NanoAssemblr system in our laboratory, and the other is called DOTAP-Lipo, which is a commonly used reagent for transfecting nucleic acids and is commercially available from Sigma in a ready-to-use format. Interestingly, T-cell responses against HEL antigen were not observed in this case even when using DOTAP-Nano plus D35. We found that the preparation method, size, and interaction with antigen are important factors influencing the adjuvanticity of DOTAP-based lipid particle adjuvant. The data are presented as mean SD, and unpaired t-test or one-way analysis of variance was used for statistical analysis. DOTAP-film was formed by the conventional lipid film hydration method, and it was directly compared with DOTAP-Nano prepared with NanoAssemblr. To induce antigen-specific T-cell responses, particularly by using protein antigen, there is a need to include an appropriate immune adjuvant. Mice were purchased from CLEA Japan. Further investigations revealed that the size of DOTAP nanoparticles, prepared buffer conditions, and physicochemical interaction with vaccine antigen are important factors for the efficient induction of T-cell responses with a relatively small antigen dose. Funding: This research was supported by KAKENHI (16K01436) and Research Foundation for Microbial Diseases of Osaka University. The bar graph indicates the mean SD of three mice per group. All rights reserved To further clarify whether these interactions are physically stable, OVA or HEL was mixed with DOTAP-Nano, and then free antigen and lipid/antigen complex were separated by UF. We then investigated the mechanisms underlying this difference in adjuvant effect. Writing review & editing, Roles Various analogues of TAP are available for structure-activity relationship studies. (C) OVA or HEL was mixed with DOTAP-Nano under Glu/PBS buffer conditions. This result shows that particle size is an important factor for the adjuvanticity of DOTAP particles. Each dot indicates one measurement. detailed instructions for the use of its reagent, the following general steps are used for essentially all formulations; times, After seven days, splenocytes were collected and stimulated with OVA257-264 peptide and OVA protein. Vaccine adjuvants generally provide two functions, namely, immune potentiator and delivery, and many adjuvants that can efficiently induce T-cell responses are known to have the combination of these two functions. After drying to form a thin lipid film on the bottom of a round-bottomed flask, the lipid film was hydrated in 5% glucose, freezed/thawed five times and then sonicated once. Methods Mol Biol. The following volumes are sufficient for transfecting a 35-mm dish or one For three times immunization experiment, the mice were immunized at the tail base with two weeks intervals such as immunization at day 0, day14, and day 28, and then the mice were sacrificed 7 days later of the last immunization such as day 35 for T cell and antibody response assays. The bar graph indicates the mean SD of three mice per group. homemade transfection reagents by adding equimolar amounts of each lipid suspended in chloroform, mixing, and drying under PMID: 15638468. The DOTAP-Nano mixtures were immediately dialyzed (50 kDa MWCO dialysis tubing; Repligen Corporation, MA) against 5% glucose solution to remove ethanol. We chose D35, K3, C2395, and P21889 as representatives of each type of CpG ODN. Furthermore, other factors such as interaction with antigen (Fig 2A and 2B) and differences in buffer conditions (Fig 1E; right) were also important. Mouse IFN-/ (type I IFN) production was measured using B16-Blue IFN-/ reporter cells (InvivoGen). D35 (1 M = 6.3 g/mL) and DOTAP-Nano/-Lipo or D35+DOTAP-Nano/-Lipo were added to the cell cultures overnight at 37C in a CO2 incubator. OVA immunization with DOTAP-Lipo plus D35 (in MES-buffered saline; under these conditions, DOTAP does not interact with OVA, as shown in Fig 1) did not induce T-cell responses compared with DOTAP-Nano plus D35 (in Glu/PBS buffer; interacting with OVA) (Fig 4A). In Glu/PBS, both DOTAP-Nano and DOTAP-Lipo showed apparent interactions with OVA, but this was not the case in the MES-buffered saline (Fig 1D). 2013;1025:269-79. doi: 10.1007/978-1-62703-462-3_21. To examine the effects of these differences in buffer conditions on the DOTAPantigen interaction in detail, we prepared DOTAP-Nano in MES-buffered saline instead of 5% glucose solution and similarly exchanged the buffer of DOTAP-Lipo from MES-buffered saline to Glu/PBS by UF. Significant differences are indicated with asterisks (*P < 0.05 and **P < 0.01). We found that the microfluidic preparation of DOTAP nanoparticles induced stronger CD4+ and CD8+ T-cell responses than liposomal DOTAP. However, the D35-mediated induction of IFN- still did not result in the strong induction of a T-cell response in the case of DOTAP-Lipo adjuvant (in MES-buffered saline; not interacting with OVA); this suggested that the interaction between DOTAP and antigen is essential even when D35 is included in DOTAP particle adjuvant. Each dot indicates one measurement. Seven days after the last immunization, splenocytes were collected and stimulated with OVA257-264 peptide and OVA protein for CD8+ and CD4+ T-cell responses, respectively. Each dot indicates one measurement. Increased amounts of antigen and adjuvant resulted in better CD8+ T-cell responses (Fig 6A; left). For more information about PLOS Subject Areas, click These results suggested that DOTAP-Nano plus D35 adjuvanticity was completely dependent on the presence of a physically stable electrostatic interaction with antigen protein. Briefly, DOTAP was dissolved in ethanol. The lipid solution (10 mg/mL) in ethanol and 25 mM acetate buffer (pH 4.0) were injected into the microfluidic mixer at a 1:3 volume and at a combined final flow rate of 15 mL/min (3.75 mL/min ethanol, 11.25 mL/min aqueous). Yes Among these antibody responses, IgG2c responses required relatively strong antigen and adjuvant dose immunization. Currently, experiments examining the biodistribution in tissue, such as in draining lymph nodes, are ongoing.
Resources, Supervision, Is the Subject Area "Immunologic adjuvants" applicable to this article? No, Is the Subject Area "Antigens" applicable to this article? In the case of CD4+ T-cell responses, MES-buffer DOTAP-Nano showed slight reductions in CD4+ T-cell responses; however, a comparable amount of IFN- production was still detected as that in Glu/PBS (Fig 1E; right). DOTAP is one of the most popularly used cationic lipid for vaccine formulation and development studies ([1821, 2427]. Twenty-four hours later, mouse IFN-gamma in the culture supernatant was determined by ELISA. Writing review & editing, Affiliation For more information about PLOS Subject Areas, click For product pricing and order placement, please click on the appropriate Location Box below. The authors would like to thank Yoshihiko Tanimoto and Mie Suzuki for useful discussions, and Saiko Ito for secretarial assistance. In this study, we explored a cationic lipid DOTAP-based adjuvant. Pezzoli D, Kajaste-Rudnitski A, Chiesa R, Candiani G. Lipid-based nanoparticles as nonviral gene delivery vectors. After seven days, splenocytes were stimulated with OVA257-264 peptide, which induced an MHC class Irestricted CD8+ T-cell response, and with OVA protein, which induced an MHC class IIrestricted CD4+ T-cell response. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, Corrections, Expressions of Concern, and Retractions, https://doi.org/10.1371/journal.pone.0227891, https://doi.org/10.1016/j.ces.2010.08.039. Low-endotoxin ovalbumin (OVA) was purchased from WAKO. The mice were sacrificed by cervical dislocation, and their spleens were collected; single-cell suspensions were prepared. Citation: Haseda Y, Munakata L, Meng J, Suzuki R, Aoshi T (2020) Microfluidic-prepared DOTAP nanoparticles induce strong T-cell responses in mice. HEL plus Freunds complete adjuvant immunization induced IFN- responses, whereas HEL with DOTAP-Nano plus D35 did not induce CD4+ T-cell responses (Fig 5A).
To further investigate the necessity of antigenlipid interaction, we tested HEL as another model antigen. Simberg D, Weisman S, Talmon Y, Barenholz Y. DOTAP (and other cationic lipids): chemistry, biophysics, and transfection. Methodology, Twenty-four hours later, the mouse IFN/ concentration in the supernatant was measured by B16-Blue reporter cell assay. The bar graph indicates the mean SD of three mice per group. DOTAP was dissolved in chloroform (510 mg/mL). Copyright 2022 by Cold Spring Harbor Laboratory Press. Human IFN- was measured with human IFN- pan-ELISA development kit (Mabtech). Hen egg lysozyme (HEL) was purchased from Sigma. The femurs and tibiae of mice (total three mice for this BM experiment) were removed, and the surrounding muscle tissues were cut using scissors. First, we compared two formulations of cationic lipid DOTAP for the T-cell-inducing adjuvant activity against model protein antigen OVA in mice. These results suggested that DOTAP-Nano plus D35 efficiently induced T-cell responses in mice even with a relatively small amount of antigen such as 1 g of OVA and only a single immunization. OVA257-264 peptidespecific MHC class Irestricted CD8+ T-cell responses were induced in a manner that is dependent on both antigen and adjuvant (DOTAP-Nano plus D35). Data curation, (205) 663-2494 Headquarters | In the case of OVA mixed with DOTAP-Nano, no free OVA was detected in the FT solution (left graph). Birmingham, AL 35243. We also demonstrated that the combination of DOTAP and Type-A CpG ODN is a simple and promising adjuvant for efficiently inducing both CD4+ and CD8+ T-cell responses against protein antigen. PBMCs were prepared from healthy Japanese adult volunteers who had provided written informed consent to participate in this study. Immune potentiators generally stimulate various innate immune pattern recognition receptors. Splenocytes were plated on 96-well flat plates at 2 106 cells/well/200 L. These results suggested that the difference in adjuvanticity of the two DOTAP preparations arose from the difference in physical interaction between antigen and cationic lipid. Methodology, Terms of Service. The mixtures were ultrafiltrated to separate lipid binding antigen and free antigen in the flow through (FT). Conceptualization, | Website by Infomedia, Headquarters |
https://doi.org/10.1371/journal.pone.0227891.g005. Although the underlying detailed mechanisms need to be examined extensively in future works, the relatively small DOTAP nanoparticle preparations obtained using a microfluidic processor such as NanoAssemblr showed better adjuvant activity than those obtained using conventional particle preparation, such as the hydration film method. BALB/c mice were similarly immunized with HEL with DOTAP-Nano plus D35 in Glu/PBS, and CD4+ T-cell responses against HEL107-116/I-Ed were evaluated by IFN- ELISA. Although many other factors can affect antigenlipid physical interactions, the electrostatic interaction was expected to be the main factor because the isoelectric point of OVA is approximately 4.5 and because DOTAP is always cationic within a broad pH range. The zeta potential of DOTAP-Nano in 5% glucose was approximately 200 mV (Fig 1C). Twenty-four hours later, mouse IFN-gamma in the culture supernatant was measured by ELISA. Thank you for your continued business and support of Avanti Polar Lipids, Inc! We chose DOTAP-Nano plus D35 adjuvant in this experiment and examined the combination of different OVA antigen doses (1, 10, and 100 g) and different D35 doses (1, 10, 20, and 30 g; notably, the ratio of D35:DOTAP weights was kept at 1:10, and DOTAP was proportionally increased as the amount of D35 increased) for the induction of T-cell responses. Vaccine Dynamics Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, No, Is the Subject Area "T cells" applicable to this article? Taken together, these results suggested that DOTAP-Nano plus D35 is a highly efficient adjuvant for inducing both CD8+ and CD4+ T-cell responses with a relatively small amount of antigen and that it is capable of inducing efficient antibody responses with the prime-boost protocol. Therefore, future experiments should be performed to understand the mechanism of action of DOTAP-Nano adjuvant. (A) C57BL/6J mice were immunized with OVA (10 g) and DOTAP (100 g) with D35 at the tail base. DOTAP-Nano was then concentrated to approximately 1.52.0 mg/mL DOTAP by using Amicon Ultra Centrifugal filters (100 kDa MWCO; Merck KGaA, Darmstadt, Germany) and sterilized through a 0.22 m PVDF filter (Merck KGaA). Mice were housed in a room maintained at constant room temperature (2224C) with a 12-hour-light/12-hour-dark cycle (lights on at 8:00 am, lights off at 8:00 pm) and had free access to food and water. DOTAP-Nano/OVA plus other types of CpG including K3, C2395, and P21889 were relatively ineffective (Fig 3). (C) Serum was collected seven days after the last immunization from C57BL/6J mice, as shown in Fig 6B. Formal analysis, DOTAP-Nano and DOTAP-Lipo only were also included as controls. https://doi.org/10.1371/journal.pone.0227891.g006. broad scope, and wide readership a perfect fit for your research every time.
Ninety-six-well plates were coated with 100 g/ml OVA or standard antibodies (total IgG: MBL, IgG1, IgG2c; Southern Biotech) in a carbonate buffer (pH 9.6). The efficient delivery of exogenous DNA to cells for expression and function studies is an essential technique of modern cell In this study, we examined the adjuvanticity of DOTAP nanoparticles, particularly for the induction of T-cell responses. Writing review & editing, Affiliations Data Availability: All relevant data are within the paper and its Supporting Information files. 
