Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.
Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.
Infectious diseases have represented a severe threat to millions of human beings around the globe and are still one of the leading causes of death in some developing countries. Prophylactic vaccination has been one of the most effective interventions of modern society to prevent and control infectious diseases1,2. These critical milestones of science in 20th-century relevance have been remarked by the recent worldwide Covid-19 pandemic caused by the SARS-CoV-2 virus3. Recognizing the importance of having efficient vaccines to curtail the dissemination of the disease, cooperative efforts from all biomedical communities have successfully resulted in many prophylactic vaccines in the market in less than a year4.
Traditionally, vaccines were composed of attenuate (live, reduced virulence) or inactivated (death particles) viruses. However, for some diseases with no margin for safety errors, viral particles are not possible, and protein subunits are used instead. Nevertheless, subunits usually do not enable the combination of more than one epitope/antigen, and adjuvants are required to enhance vaccination potency5,6. Therefore, the need for novel vaccine types stands clear.
As demonstrated during the current pandemic, novel vaccine candidates based on nucleic acids can be advantageous in terms of avoiding long development processes and providing high versatility while producing, at the same time, a vital patient immunization. This is the case of mRNA vaccines, which were initially designed as experimental cancer vaccines. Thanks to their natural capacity to produce antigen-specific T-cell responses3,5,6,7. Being mRNA the molecule that encodes the antigenic protein, only changing the same, the vaccine can be rapidly tailored to immunize other variants of the same microorganism, different strains, other infectious microorganisms, or even become a cancer immunotherapeutic treatment. In addition, they are advantageous in terms of large-scale production costs. However, mRNA has a significant hurdle that hampers their naked administration: its stability and integrity are compromised in physiological media, full of nucleases. For this reason, the use of a nanometric carrier that protects it and vectorizes mRNA to the antigen-presenting cells is required2,8.
In this context, poly(beta aminoesters) (pBAE) are a class of biocompatible and biodegradable polymers that demonstrated a remarkable ability to complex mRNA in nanometric particles, thanks to their cationic charges9,10,11. These polymers are composed of ester bonds, which makes their degradation easy by esterases in physiological conditions. Among the pBAE library candidates, those functionalized with end cationic oligopeptides showed a higher capacity to form small nanoparticles to efficiently penetrate cells through endocytosis and transfect the encapsulated gene material. Furthermore, thanks to their buffering capacity, the acidification of the endosome compartment allows endosomal escape12,13. Namely, a specific kind of pBAE, including hydrophobic moieties on their backbone (the so-named C6 pBAE) to enhance their stability and end-oligopeptide combination (60% of polymer modified with a tri-lysine and 40% of the polymer with a tri-histidine) that selectively transfects antigen-presenting cells after parenteral administration and produce the mRNA encoded antigen presentation followed by mice immunization has been recently published14. In addition, it has also been demonstrated that these formulations could circumvent one of the main bottleneck steps of nanomedicine formulations: the possibility to freeze-dry them without losing their functionality, which enables long-term stability in soft dry environments15.
In this context, the objective of the current protocol is to make the procedure for the formation of the mRNA nanoparticles available to the scientific community by giving a description of the critical steps in the protocol and enabling the production of efficient vaccines for infectious diseases prevention and tumor treatment applications.
The following protocol describes the complete workout to synthesize oligopeptide end-modified poly(beta aminoesters) – OM-pBAE polymers that will further be used for nanoparticle synthesis. In the protocol, nanoparticles formulation is also included. In addition, critical steps for the success of the procedure and representative results are also provided to ensure that the resulting formulations accomplish the required quality control characterization features to define a positive or negative result. This protocol is summarized in Figure 1.
1. Synthesis of pBAE polymer with end oligopeptides (OM-pBAE)
2. Polyplexes formation
NOTE: All the procedures should be performed inside a conditioned room to maintain a constant temperature.
3. Polyplexes lyophilization
4. Polyplex resuspension
NOTE: This protocol describes the process used to reconstruct the lyophilized C6-peptide-pBAE nanoparticles for their further use either for characterization, in vitro, or in vivo analysis.
5. Polyplex characterization
6. In vitro characterization
7. In vitro functionality tests: capacity to activate model immune cells by using ovalbumin (OVA) as antigenic model mRNA
Polymer synthesis and characterization
The OM-pBAE synthesis procedure is given in Figure 2. As Figure 2A shows, the first step to obtain the OM-pBAE is to synthesize the C6-pBAE by adding the amines (1-hexylamine and 5-amino-1-pentanol, ratio 1:1) to the diacrylate (1,4-butanediol diacrylate). This reaction is carried out at 90 °C for 20 h and with constant stirring. Afterward, a solution of oligopeptides is added to a solution of C6 polymer obtained from the previous reaction (Figure 2B). Finally, the synthesis is carried out using DMSO as a solvent at 25 °C with continuous stirring for 20 h. Figure 2C shows the structure of the OM-pBAE, which is the resulting product of polymerization. The resulting polymers are characterized by 1H-NMR (see Supplementary Figure S2). In the characterization, a positive result is the one with several monomer units (n + m in the figures) comprised between 7-10; distributed half one unit type and half the other; while any other number outside this range is a negative result.
Nanoparticle synthesis and physicochemical characterization
Table 3 shows the results of the physicochemical characterization of the GFP mRNA encapsulating nanoparticles as a proof of concept. It was previously demonstrated that size and surface charge do not show significant differences regardless of the mRNA type encapsulated14. Characterization has been performed both in freshly prepared and lyophilized nanoparticles to prove the stability of their physicochemical properties and to demonstrate that, as previously published, the freeze-drying process was adjusted to these formulations15. The hydrodynamic radius of the polyplexes is considered correct when it ranges around 150-220 nm. Besides, the PDI must be constant, approximately 0.2, but it is accepted up to 0.3. Therefore, neither size nor PDI showed significant differences before and after lyophilization, as shown here using representative results. Also, nanoparticle sizing was performed with NTA, to confirm the previously obtained results. Any value outside the ranges specified above is considered a negative result, and particles should be discarded for further use.
During the physicochemical characterization of this type of sample, it is convenient to compare the results obtained by DLS with the results obtained by NTA. Both techniques determine the hydrodynamic radius of the particles by measuring the diffusion coefficient. The number of nanoparticles counted per frame in NTA is between 80 and 100 to accurately track the particles. Surface charge was also characterized to ensure the positive charge of the nanoparticles, facilitating electrostatic interaction with the cell membrane. Finally, the morphology of the polyplexes was characterized by Transmission Electronic Microscopy (TEM). Figure 3 confirms the formation of spherical and monodisperse nanoparticles of an approximate size of 50 nm diameter. Smaller diameters were obtained by analyzing TEM images as both NTA and DLS measure the hydrodynamic diameters. Although a smaller size is always expected when performing electron microscopy measurements16,17, as compared to scattering measurements, it is essential to highlight here that the difference is almost 100 nm, which is usually not expected. This is attributed to the high hygroscopic character of these nanoparticles, given by both the encapsulated mRNA and the end terminal oligopeptides composing the polymer.
mRNA encapsulation efficiency determination
Another critical parameter to measure is the ability of the nanoparticles to encapsulate the mRNA, which is the active principle and needs to be encapsulated to achieve its protection from nucelases18,19,20. The protocol has been developed to analyze the encapsulation efficiency (EE; in %) of nucleic acid entrapment following the supplier's instructions. The obtained value gives an idea about the nucleic acid percentage that is successfully entrapped into the nanoparticles. RiboGreen dye, a fluorescent nucleic acid stain, is used for this purpose. The protocol consists of quantifying any RNA that remains accessible to the fluorescent dye. To quantify the total RNA, the nanoparticles need to be disassembled, and Heparin is used for this purpose.
Table 4 shows the encapsulation efficiency (EE%) of the OM-pBAE polyplexes encapsulating both GFP and OVA mRNA, as proof of concept. RiboGreen fluorescence assay allows calculating the amount of mRNA entrapped in the nanoparticles after disassembly by heparin addition to release their content. The efficiency values obtained must be higher than 80% and, as demonstrated here, do not show significant differences depending on the type of mRNA. Lower encapsulation efficiency values represent a negative result and constitute a signal for discarding these formulations. These results confirm the high encapsulation capacity of the mRNA of OM-pBAE, its versatility, and its promising application in gene delivery.
In vitro representative results
It is essential to determine the capacity of pBAEs polyplexes. First, to transfect and, second, to activate immune cells14. Transfection efficiency is assessed by two different techniques, using eGFP as the reporter mRNA: fluorescence microscopy to visually determine the reporter protein and flow cytometry expression to quantify the transfection efficiency. A fluorescent microscope equipped with a CCD camera, 5x, 10x, 20x, and 40x objectives and UV, blue and green filter lasers were employed to qualitatively determine the expression of eGFP protein in JAWSII cell line, a murine dendritic cell line. eGFP protein has an excitation maximum at 488 nm, corresponding to blue laser, and an emission maximum around 510 nm, corresponding to green color. To quantify the transfection efficiency expression of the target protein, flow cytometry analysis of the transfected cells is performed after 24 h of the transfection with pBAEs polyplexes with eGFP mRNA. A fixation step is helpful when cells have been transfected with fluorescent reporters such as GFP or RFP.
Figure 4 shows the qualitative analysis of the eGFP expression after 24 h of transfection in JAWSII cell line. Again, compared to the negative control, the eGFP reporter signal is significatively higher. Also, Table 5 shows the transfection efficiency displayed by OM-pBAE nanoparticles in JAWSII cell line. Again, the efficiency values are comparable to positive control performed with a classical transfection reagent.
The capacity of pBAEs NPs loaded with OVA mRNA to activate model immune cells has been assessed transfecting JAWSII cell line, a murine immature dendritic cell line. To determine the activation of the immune cells, two different markers: CD11b and CD86 were employed. First, select the proper fluorophores for the flow cytometer configuration. Here, PerCP-Cy5.5 (CD11b) and PE (CD86) are used. Immature and non-activated cell lines must show a CD11b-/CD86- profile, whereas activated cells must present a CD11b+/CD86+ profile. The blue laser at 488 nm is used to determine the target protein expression, and an anti-OVA antibody plus a secondary anti-mouse-Alexa488 antibody was employed. Table 6 shows the final panel employed in this experiment. Figure 5 shows the capacity of OVA mRNA-loaded OM-pBAE nanoparticles to activate DCs, which is indicative of the activation of the cellular immune response. Non-transfected cells show a low number of cells expressing the CD11b and CD86 membrane markers, whereas the mRNA OVA OM-pBAEs treated cells show a significatively increased expression of the CD11b and CD86 markers. This result suggests that OM-pBAE nanoparticles promote maturation. Altogether, these results confirm the capacity of OM-pBAEs nanoparticles to efficiently transfect dendritic cells and mediate the activation of the immune response promoting the maturation of immature DCs.
Figure 1: Schematic representation of the whole synthetic protocol. In this figure, the mRNA vaccine synthesis steps are detailed, including the critical characterization parameters. Please click here to view a larger version of this figure.
Figure 2: Synthesis of C6-pBAE oligopeptide end-modified (OM-pBAE). (A) Raw chemicals to perform the Michael addition. (B) pBAE + oligopeptide. (C) OM-pBAE. R can be Histidine (H) or Lysine (K). Please click here to view a larger version of this figure.
Figure 3: TEM images of fresh GFP mRNA encapsulating OM-pBAE nanoparticles. Polyplexes were negatively stained for electronic microscopy characterization. Monodisperse particles of approximately 60 nm diameter are shown. (A) and (B) images represent two different microscopies of the same sample. Please click here to view a larger version of this figure.
Figure 4: Fluorescence microscopy imaging of JAWSII transfected cells with OM-pBAE/eGFP mRNA nanoparticles. (A) Bright field image. (B) GFP fluorescence (in green). (C) Composite of the previous two images. Scale bars = 100 mm. Please click here to view a larger version of this figure.
Figure 5: Activation of JAWSII after transfection with OM-pBAE/OVA mRNA nanoparticles. Negative control (CN) values correspond to the black label. Transfected cells correspond to the gray label. Bars correspond to the standard deviation (SD) of three replicates. Please click here to view a larger version of this figure.
Material | Material | Microvesicles |
Refractive Index | 1.4 | |
Absorption | 0.01 | |
Dispersant | Dispersant | Water |
Temperature (°C) | 25 | |
Viscosity (cP) | 0.8872 | |
Refractive Index | 1.33 | |
Temperature | Temperature (°C) | 25 |
Equilibration time (s) | 10 | |
Cell | Cell type | ZEN0040 |
Table 1: Parameters to create an SOP for size measurements.
Material | Material | Microvesicles |
Refractive index | 1.4 | |
Absortion | 0.01 | |
Dispersant | Dispersant | Water |
Temperature (°C) | 25 | |
Viscosity (cP) | 0.8872 | |
Refractive index | 1.33 | |
Temperature | Temperature (°C) | 25 |
Equilibration time | 10 | |
Cell | Cell type | DTS1070 |
Table 2: Parameters to create an SOP for zeta potential measurements.
GFP mRNA/OM-pBAE | T (oC) | Hydrodinamic diameter (nm) | PDI | Surface charge (mV) | |
NTA | DLS | ||||
Fresh | 25 | 124 ± 32 | 134 ± 8 | 0.2 ± 0.04 | 32 ± 0.7 |
Lyophilized | 25 | 135 ± 35 | 138 ± 2 | 0.17 ± 0.02 | 34 ± 0.7 |
Table 3: Physicochemical characterization of mRNA encapsulating OM-pBAE nanoparticles. GFP mRNA encapsulating OM-pBAE polyplexes were characterized immediately after preparation – fresh- and after lyophilization process to ensure the stability of the particles. DLS and NTA sizing, and surface charge data are shown.
EE% | |
GFP mRNA/OM-pBAE | 82.3 ± 1.5 |
OVA mRNA/OM-pBAE | 83.1 ± 1.5 |
Table 4: Encapsulation efficiency (EE%) data of mRNA encapsulating OM-pBAE nanoparticles. Encapsulation efficiency percentages of GFP and OVA mRNA encapsulating OM-pBAE nanoparticles. RiboGreenfluorescence assay was performed on lyophilized samples to assess the amount of mRNA entrapped in the polyplexes.
Sample | % GFP positive cells | SD |
Negative Control | 1.85% | 0.21% |
OM-pBAE/eGFP mRNA transfected cells | 57.79% | 5.28% |
Table 5: Transfection efficiency of OM-pBAE/eGFP mRNA nanoparticles in JAWSII cell line. Transfection efficiency was tracked by flow cytometry.
Antibody | Cells stained | Fluorescent label |
CD11b | Activated DCs | PerCP-Cy 5.5 |
CD86 | Activated DCs | PE |
α-mouse AlexaFluor488 | OVA positive cells | AlexaFluor 488 |
Table 6: Flow cytometry panel. This panel was used for flow cytometry studies to determine the in vitro functionality of the mRNA vaccine.
Supplementary information: See the Supplemental File with all Supplementary information included. Please click here to download below Files.
Table S1A: Ribosomal RNA standard.
Table S1B: RNA:pBAE with heparin standard.
Table S1C: Heparin standard.
Figure S2: 1H-NMR proton spectra of C6-pBAE and OM-pBAE. (A) 1H-NMR from the C6-pBAE. Chloroform-d was used as solvent (δ = 7.25 ppm). (B) 1H-NMR from the C6CH3. D2O was used as solvent (δ = 4.64 ppm).
After the outbreak of the Covid-19 pandemic last year, the importance of vaccines in terms of infectious disease control has manifested as a critical component8. Efforts from scientists worldwide have enabled the release to the market of many vaccines. For the first time in history, mRNA vaccines have demonstrated their previously hypothesized success, thanks to their rapid design because of their capacity to adapt to any novel antigen within some months5,6,21. mRNA, or messenger ribonucleic acid, refers to the nucleic acids that drive protein synthesis from encoded information in the DNA. In here, for vaccination purposes, the mRNA codifies for an antigenic protein belonging to the infectious microorganism (or to a tumor antigen in case of therapeutic tumor vaccination)8,22. In this context, it is critical for the scientific community to have clear protocols for synthesizing mRNA vaccines.
With this idea in mind, a straightforward procedure for synthesizing mRNA vaccines based on the proprietary polymeric nanoparticles was aimed to be described24. As described in the protocol section, first, the polymers are synthesized using a two-step procedure based on a Michael addition of primary amines to acrylate groups to synthesize polymer backbones, followed by the addition of terminal oligopeptides to give the enhanced cationic character and capacity to escape from endosomes to the resulting polymers12,13,14. Then, in a further step, nanoparticles are prepared by mixing the polymers with the mRNA to achieve their electrostatic interaction to form small nanometric particles.
Although the synthesis of the polymers is a straightforward protocol, the same cannot be said for the nanoparticle formulation. Many collaborators worldwide have used these polymers and have found common critical steps when referring to the preparation and use of the particles. Taking into account that the synthesis is by manual mixing, the user abilities play a crucial role. The trickiest step is mixing the polymer and the mRNA, which must take place in a controlled way, as it must be done for the further precipitation step of the mixture to the water and the buffer addition. Any modification on the mechanics of mixing the two components will result in microparticles (instead of nanoparticles), which cannot be used for humans. Therefore, mixing is one of the troubleshooting steps that needs some practice to be achieved correctly.
Another striking point is freeze-drying. As it is widely known, it represents one of the bottleneck steps of nanoformulations. In this specific case, it took some years to adjust all the parameters and describe a protocol that succeeded15. Even having that adjusted, another critical step of the protocol is the redispersion of the formulations, which, if not performed correctly, bring nanoparticles' aggregation and they lose their functionality. In this case, it is a must to rapidly place the formulations in a dry, slightly cold environment to avoid their uncontrolled rehydration. Due to their high hygroscopic nature, special attention must be put to rehydrate them in a controlled way by carefully pipetting up and down all the sticky powder formed by lyophilization. If the samples rehydrate in a non-controlled way, they will immediately aggregate forming a translucid to opaque dispersion.
Although the critical steps are mentioned here, the protocol for synthesizing pBAE polyplexes is easy and rapid, which is advantageous among other synthesis methods of the gene delivery systems. Here, the specific application of mRNA vaccination has been provided in detail. However, it represents a versatile methodology that can also be applied to encapsulate other types of nucleic acids and non-vaccine use, such as in oncology and cardiovascular fields, as published previously13,23,24.
In conclusion, this protocol will enable these proprietary polymers to be available to the scientific community to design novel mRNA vaccines while avoiding all this troubleshooting. These polymers are advantageous compared to other mRNA lipid formulations regarding the possibility of freeze-drying, long-term stability, and decrease of storage and distribution costs by not requiring ultra-cold temperatures. Therefore, it is expected that OM-pBAE vaccines play a vital role in the vaccination field for prophylactic and therapeutic applications.
The authors have nothing to disclose.
Financial support from MINECO/FEDER (grants SAF2015-64927-C2-2-R, RTI2018-094734-B-C22, and COV20/01100) is acknowledged. CGF acknowledged her IQS PhD Fellowship.
1,4-butanediol diacrylate | Sigma Aldrich | 123048 | |
1-hexylamine | Sigma Aldrich | 219703 | |
5-amino-1-pentanol | Sigma Aldrich | 411744 | |
Acetone | Panreac | 141007 | |
CD11b antibody | BD | 550993 | |
CD86 antibody | Bioligend | 105007 | |
Chlor hydroxhyde | Panreac | 181023 | |
Chloroform-d | Sigma Aldrich | 151823 | |
Cys-His-His-His peptide | Ontores | Custom | |
Cys-Lys-Lys-Lys peptide | Ontores | Custom | |
D2O | Sigma Aldrich | 151882 | |
DEPC reagent for Rnase free water | Sigma Aldrich | D5758 | This reagent is important to treat MilliQ water to remove any RNases of the buffers |
Diethyl eter | Panreac | 212770 | |
dimethyl sulfoxide | Sigma Aldrich | 276855 | |
HEPES | Sigma Aldrich | H3375 | |
mRNA EGFP | TriLink Technologies | L-7601 | |
mRNA OVA | TriLink Technologies | L-7610 | |
RiboGreen kit | ThermoFisher | R11490 | |
sodium acetate | Sigma Aldrich | 71196 | |
sucrose | Sigma Aldrich | S0389 | |
Trifluoroacetic acid | Sigma Aldrich | 302031 | |
Trypsin-EDTA | Fisher Scientific | 11570626 | |
α-mouse AlexaFluor488 antibody | Abcam | Ab450105 | |
Equipment | |||
Nanoparticle Tracking Analyzer | Malvern Panalytical | NanoSight NS300 | |
Nuclear Magnetic Ressonance Spectrometer | Varian | 400 MHz | |
ZetaSizer | Malvern Panalytical | Nano ZS | For zeta potential and hydrodynamic size determination |
Software | |||
NanoSight NTA software | Malvern Panalytical | MAN0515-02-EN-00 | |
NovoExpress Software | Agilent | Not specified | |
ZetaSizer software | Malvern Panalytical | DTS Application | To analyze surface charge and hydrodynamic sizes |