cancers

Article
Intratumoural Delivery of mRNA Loaded on a Cationic
Hyper-Branched Cyclodextrin-Based Polymer Induced
an Anti-Tumour Immunological Response in Melanoma
Yousef Khazaei Monfared 1,† , Mohammad Mahmoudian 2 , Parvin Zakeri-Milani 2, *, Claudio Cecone 1 ,
Tomoya Hayashi 3 , Ken J. Ishii 3 , João Conde 4 , Adrián Matencio 1 and Francesco Trotta 1, *,†

1 Department of Chemistry, University of Turin, 10125 Turin, Italy; yousef.khazaeimonfared@unito.it (Y.K.M.);

claudio.cecone@unito.it (C.C.); adrian.matencioduran@unito.it (A.M.)
2 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5165665931, Iran;
mahmoodian.nano@gmail.com
3 Division of Vaccine Science, Department of Microbiology and Immunology, The Institute of Medical Science,
The University of Tokyo (IMSUT), Tokyo 113-8654, Japan; tomoya-h@ims.u-tokyo.ac.jp (T.H.);
kenishii@ims.u-tokyo.ac.jp (K.J.I.)
4 ToxOmics, NOVA Medical School (NMS), Faculdade de Ciências Médicas (FCM), Universidade Nova de Lisboa,
1099-085 Lisboa, Portugal; joao.conde@nms.unl.pt
* Correspondence: pzakeri@tbzmed.ac.ir (P.Z.-M.); francesco.trotta@unito.it (F.T.)
† These authors contributed equally to this work.

Simple Summary: The frequency of metastatic melanoma, an extremely deadly malignancy, is

rapidly increasing worldwide. Recently, messenger RNA (mRNA) injections have emerged as
a promising treatment option. While mRNA therapies have demonstrated significant promise,
the stability of the naked form remains a barrier, as naked mRNAs are vulnerable to common
ribonucleases, and are unable to effectively penetrate plasma membranes and escape from endosomes.
Thus, for this paper, we used a hyper-branched cyclodextrin-based polymer (Ppoly) as a carrier to
Citation: Khazaei Monfared, Y.;
Mahmoudian, M.; Zakeri-Milani, P.;
enhance mRNA delivery for melanoma cancer. The in vitro results demonstrated that Ppoly was able
Cecone, C.; Hayashi, T.; Ishii, K.J.; to deliver the EGFP-mRNA effectively in both 2D and 3D melanoma cell lines compared to naked
Conde, J.; Matencio, A.; Trotta, F. mRNA; in addition, Ppoly did not show any cytotoxicity. The anti-tumour effect of intratumourally
Intratumoural Delivery of mRNA injected OVA-mRNA loaded on Ppoly results showed a significant decrease in both tumour size
Loaded on a Cationic Hyper-Branched and weight compared to other formulations by inducing an efficient adaptive immune response and
Cyclodextrin-Based Polymer Induced OVA-specific CD8+ T cells in both spleen and tumour tissues compared to other groups.
an Anti-Tumour Immunological
Response in Melanoma. Cancers 2023, Abstract: mRNA technology has demonstrated potential for use as an effective cancer immunother-
15, 3748. https://doi.org/10.3390/ apy. However, inefficient in vivo mRNA delivery and the requirements for immune co-stimulation
cancers15143748
present major hurdles to achieving anti-tumour therapeutic efficacy. Therefore, we used a cationic
Academic Editors: Antonio Curti and hyper-branched cyclodextrin-based polymer to increase mRNA delivery in both in vitro and in vivo
Adam C. Berger melanoma cancer. We found that the transfection efficacy of the mRNA-EGFP-loaded Ppoly system
was significantly higher than that of lipofectamine and free mRNA in both 2D and 3D melanoma
Received: 24 June 2023
cancer cells; also, this delivery system did not show cytotoxicity. In addition, the biodistribution
Revised: 19 July 2023
Accepted: 20 July 2023
results revealed time-dependent and significantly higher mEGFP expression in complexes with Ppoly
Published: 24 July 2023 compared to free mRNA. We then checked the anti-tumour effect of intratumourally injected free
mRNA–OVA, a foreign antigen, and loaded Ppoly; the results showed a considerable decrease in both
tumour size and weight in the group treated with OVA-mRNA in loaded Ppoly compared to other
formulations with an efficient adaptive immune response by dramatically increasing most leukocyte
Copyright: © 2023 by the authors. subtypes and OVA-specific CD8+ T cells in both the spleen and tumour tissues. Collectively, our
Licensee MDPI, Basel, Switzerland.
findings suggest that the local delivery of cationic cyclodextrin-based polymer complexes containing
This article is an open access article
foreign mRNA antigens might be a good and reliable concept for cancer immunotherapy.
distributed under the terms and
conditions of the Creative Commons
Keywords: cyclodextrin-based polymer; mRNA delivery; melanoma cancer; immunotherapy
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Cancers 2023, 15, 3748. https://doi.org/10.3390/cancers15143748 https://www.mdpi.com/journal/cancers

Cancers 2023, 15, 3748 2 of 19

1. Introduction
The incidence of metastatic melanoma, an extremely aggressive and fatal cancer, is
steadily increasing worldwide. Early detection and the surgical removal of tumours are
crucial to prevent its spread and potentially fatal outcomes. Conventional treatments like
irradiation and chemotherapy have shown limited success [1]. However, new treatments
focusing on oncogenic drivers have made some progress. Recently, messenger RNA
(mRNA) injections have emerged as one such treatment that provides low mutational risk,
good safety, and has the adaptive modularity to express a variety of diverse therapeutically
useful proteins [2–5]. Research in molecular medicine is now focusing on mRNA. This
is particularly true when it comes to immunisation. Many mRNA vaccines, including
those for cancer immunotherapy and those against viral infections, have entered clinical
trials [6,7]. The success of mRNA-based therapy has been demonstrated in numerous
applications, including gene editing, protein replacement therapy, cancer immunotherapy,
and immunisation against the SARS-CoV-2 virus [3,6,7]. While mRNA therapies have
demonstrated significant promise, the stability of the naked form remains a barrier, as naked
mRNAs are vulnerable to common ribonucleases, unable to penetrate plasma membranes
effectively, and unable to escape from endosomes [8]. To overcome these obstacles, one
potential solution is the encapsulation of mRNA within nanoparticles. This protective
encapsulation not only shields mRNA from enzymatic degradation but also facilitates
efficient cellular uptake and transport to different regions of the body. As a result, there
has been a growing interest among research teams in the field of mRNA nanoparticle
delivery [9–11]. Recent advancements in cancer immunotherapy have been attributed to
the use of mRNA nanoparticles, which have demonstrated therapeutic efficacy in tumour
vaccination [12–14]. However, a critical challenge that remains is the need for effective
delivery methods. This underscores the importance of developing sophisticated and
targeted delivery approaches [9,15,16]. On the other hand, polymers have attracted interest
as a platform for gene delivery due to their desirable qualities, such as non-integration,
scalable manufacturing, and extensive chemical structural flexibility [17–19]. Cyclodextrin
(CD) has been recognised as an effective and efficient oligonucleotide delivery technique
among polymer delivery technologies [17,18,20]. Originally employed as a plasmid DNA
delivery vehicle in 1999, CD has since been recognised for its utility as a carrier for siRNAs
(small interfering RNAs) [21]. CD-modified polycations have demonstrated beneficial
biocompatibility and a good capacity to create stable polyplexes with therapeutic nucleic
acids to keep them stable [22]. Due to their relatively high transfection effectiveness and
modification adaptability, cyclodextrin-based nanosponges (CDNSs) and their derivatives
have also been identified as frequently used mRNA vaccine delivery systems [23–25].
Clinical evidence has highlighted the potential benefits of local immunotherapy in
eliminating the toxicities associated with systemic treatments while promoting robust
immune responses against cancer [26–28]. With this in mind, our investigation aims to
assess whether cationic hyperbranched cyclodextrin-based polymers (Ppoly), known for
their remarkable efficiency as carriers for pDNA transport in both 2D and 3D spheroid
cells without toxicity [29], can effectively overcome the challenges associated with mRNA
delivery for melanoma cancer in vivo. Therefore, in the present study, we aimed to inves-
tigate the capacity of our newly synthesised Ppoly by imparting positive charges to the
final product using choline chloride (CHO), which has been extensively studied for its
non-toxicity [30,31] and potential to be used as an effective delivery system to overcome
the challenges related to mRNA delivery in cancer immunotherapy. To test this hypothesis,
we pursued the following aims: Firstly, we checked the effect of Ppoly to enhance the trans-
fection efficiency and cellular uptake of mRNA encoding the EGFP in 2D and 3D spheroid
melanoma cancer cells (B16-F16). Then, the oval albumin (OVA)-mRNA, a foreign antigen,
was loaded on Ppoly and administered intratumourally to pulse and induce immune cell
responses in a melanoma mice model (Figure 1). The rationale behind using OVA-mRNA
for this study is based on the concept of antigen presentation and the activation of the
adaptive immune system. When the OVA-mRNA is translated to the OVA protein (a foreign
 enhance the transfection efficiency and cellular uptake of mRNA encoding the EGFP in
2D and 3D spheroid melanoma cancer cells (B16-F16). Then, the oval albumin (OVA)-
mRNA, a foreign antigen, was loaded on Ppoly and administered intratumourally to
pulse and induce immune cell responses in a melanoma mice model (Figure 1). The
Cancers 2023, 15, 3748 rationale behind using OVA-mRNA for this study is based on the concept of antigen 3 of 19
presentation and the activation of the adaptive immune system. When the OVA-mRNA is
translated to the OVA protein (a foreign protein) within the cancer, they can potentially
present fragments of those proteins on their cell surface using molecules called major
protein) within the cancer, they can potentially present fragments of those proteins on their
histocompatibility complex (MHC) molecules. These MHC molecules display the protein
cell surface using molecules called major histocompatibility complex (MHC) molecules.
fragments to immune cells, such as T cells. If the immune system recognises the displayed
These MHC molecules display the protein fragments to immune cells, such as T cells. If the
protein fragments as foreign or abnormal, it can trigger an immune response against the
immune system recognises the displayed protein fragments as foreign or abnormal, it can
cells displaying those fragments. This immune response can include the activation and
trigger an immune response against the cells displaying those fragments. This immune
proliferation of T cells, which can specifically recognise and eliminate cells presenting the
response can include the activation and proliferation of T cells, which can specifically
foreign protein fragments. Furthermore, the adjuvant ability of Ppoly was tested in
recognise and eliminate cells presenting the foreign protein fragments. Furthermore, the
healthy animals.
adjuvant ability of Ppoly was tested in healthy animals.

Figure 1.
Figure 1. Schematic rationale
rationale of
of the
the study
study design.
design.

2. Materials and Methods

2.1. Materials and Reagents
All chemical reagents were supplied by Sigma-Aldrich (St. Louis, MO, USA) without
additional purification. The OVA-modified and EGFP (5 moU) mRNAs, as well as Dream-
Fect ™ (DF40500), were provided by OZ-Biosciences (Marseille, France). Before use, β-CD
and choline chloride were dried to a constant weight in an oven at 75 ◦ C.
Cancers 2023, 15, 3748 4 of 19

2.2. Polymer Synthesis

We followed the same steps as in our past work to create the polymer [29]. In brief, the
polymer synthesis involved dissolving 1.00 g (8.81 × 10−4 mol) of anhydrous CD in 7.5 mL
of DMSO at room temperature. Following full solubilisation, 1.11 g (7.93 × 10−3 mol)
of choline chloride (CHO) and 1.14 g (7.05 × 10−3 mol) of carbonyldiimidazole (CDI)
were added. In order to separate the polymer from unreacted reactants, byproducts,
and solvent residues, the dry product was then dissolved in distilled water and filtered
using ultrafiltration (cut-off 5 kDa). The final step was to recover the product from the
ultrafiltration cell and freeze dry it to create a white powder with 22 kDa molecular weight.

2.3. Polymer–DNA Complex Formation and Gel Retardation Assay

Prior to the transfection procedures, a complex between cationic-CD polymers and
both mRNAs (EGFP and OVA) was created. Different N/P ratios of polymer/mRNA com-
plexes were formed (1:1, 5:1, and 10:1) by mixing 10 µL of mRNA solution (containing 1 µg
of EGFP and OVA mRNA in filtered distilled water) and 10 µL of polymer solution (con-
taining varying amounts of polymer in filtered distilled water). They were mixed, vortexed
for 10 s, and then incubated for 30 min at room temperature to create the complexes. Finally,
gel electrophoresis was run by mixing the loading buffer and transferring the resulting
solutions onto a 1% agarose gel in 1× TAE buffer (40 mmol/L Tris acetate and 1 mmol/L
EDTA) at 90 V for 45 min. DNA bands were observed using a UV trans-illuminator.

2.3.1. Complexes Characterisation

As previously reported, characterisation of the complexes was carried out [29]. In
brief, a 90-plus particle sizer was used to measure the size of the complexes and their zeta
potential (Malvern Instruments, Malvern, UK). Furthermore, the structural characteristics
and the presence of interactions between polymer and mRNA complexes were examined
using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy
(FTIR), respectively.

2.3.2. Determination of mRNA Encapsulation Efficiency

The ultracentrifugation approach was followed to measure the efficiency of the
nanoparticle’s encapsulation of mRNA. The difference between the total amount of mRNA
added to the buffer, which contains nanoparticles, and the amount of free mRNA left
available in the aqueous suspension was measured via nanodrop spectrophotometry at
260 nm [32].

2.4. Cell Culture Protocol for 2D and 3D Spheroid Cell Culture

B16–F10 cells (passage number: 4) were obtained from the National Cell Bank of the
Pasteur Institute, Tehran, Iran. The cell growth conditions were the same as in our previous
study [33], where 3 × 104 B16–F10 cells were used to create 3D cancer cell spheroids using
the traditional hanging drop method.

2.4.1. MTT (3-(4, 5-Dimethylthiazol-2-yl) 2, 5-Diphenyl Tetrazolium Bromide) Assay

The cytotoxicity of free-Ppoly at various concentrations was evaluated using the
MTT assay, which was slightly modified from a previous report [34]. Furthermore, the
cytotoxicity effect of different ratios (1:1, 1:5, and 1:10) of free Ppoly and complexes with
mRNA was evaluated compared to lipofectamine at concentrations of 3, 6, and 12 µL in
free and in complexes with mRNA after 24 h.

2.4.2. Measurement of EGFP (mRNA)-Polymer Transfection Using Fluorescence Microscopy

in 2D and 3D
For the 2D and 3D cell culture models, trypsinised B16–F10 cells were seeded in 12-well
plates in RPMI medium with 10% FBS at a density of 1 × 106 cells per well and 3 × 104 cells
per 20 µL drop, respectively. After 24 h, the culture medium was changed to a serum-free
Cancers 2023, 15, 3748 5 of 19

medium containing 1 and 2 µg of free mRNA-EGFP and complexed with PPoly at a N/P
ratio of 10 for 4 h for the 2D and 3D cells, respectively. The cells were then incubated for 20 h
in media containing 10% FBS. Next, PBS was used to wash the cells twice. Finally, the cells
were fixed with 4% paraformaldehyde for fluorescence microscopy observation (Cytation
5 Cell Imaging Multi-Mode Reader, Agilent, Santa Clara, CA, USA), and then nuclear
staining with 40 ,6-diamidino-2-phenylindole (DAPI) was performed at room temperature
for 30 min. In addition, the DreamFectTM Transfection Reagent (Lipofectamine), Invitrogen,
Waltham, MA, USA, was used as a positive control for 24 h on both 2D and 3D cell culture,
according to the manufacturer’s instructions. Furthermore, using flow cytometry, the same
procedure was used to quantify the cellular uptake of free and EGFP-mRNA in complexes
with PPoly at a ratio of 1:10, and Lipofectamine was used as a positive control for 24 h in
both 2D and 3D cell culture at a ratio of 3:1 (Lipofectamine/EGFP). The BD FACS Calibur
system was used to assess the cells and determine the fluorescence intensity of each group,
which was then compared to the untreated control cells. FlowJo software was used to
examine the raw data (ver. 7.6.1).

2.5. In Vivo Biodistribution of EGFP-mRNA Complexes with PPoly

All studies were performed in accordance with the National Research Council’s Guide
for the Care and Use of Laboratory Animals. The experiments were approved by the ethical
committee of Tabriz University of Medical Sciences, Pharmacy department, Iran (Ethical
Code: IR.TBZMED.AEC.1402.001). The female C57BL6/J mice were 5.5 weeks old, and
the tumours were established by injecting a suspension of B16–F10 cells (1 × 105 cells/mL)
subcutaneously (S.C.) to form the melanoma cancer [35]. Additionally, 100 µL of EGFP-
mRNA (10 µg) in complex with PPoly (containing the same quantity of free mRNA) at
ratio 1:10 was injected intratumourally when the tumours reached a volume of about
400–500 mm3 (n = 4 mice per group). The in vivo imaging system (Kodak live animal
imaging) was used to determine the quantity of fluorescence intensity at predetermined
time points.

2.6. Experimental Design and Tumour Induction

Female C57BL6/J mice aged six to eight weeks were obtained from the Zanjan Animal
Institution in Iran. B16–F10 cells (1 × 105 cells/mL) were administered subcutaneously
(S.C.) into the mice to establish melanoma cancer [35]. Sixteen mice were randomly divided
into four groups: untreated tumours, free-OVA mRNA, free-Ppoly, and OVA-mRNA in
complexes with PPoly. When the tumours appeared, the mice were given four intra-
tumoural injections of free-OVA mRNA (7 µg) in complexes with Ppoly at N/P at a ratio of
1:10, as well as the same volume of PBS for the control group. Thereafter, the mice were
sacrificed after 32 days, and tumour size was measured every day using a digital calliper.
At the end of the treatment period, the melanoma tumours were removed from the mice
and weighed. The width, length, and height of the tumours were then measured using
callipers, and the volume of the tumours was determined using the following formula:
length × width 2 × 0.52 [36].

2.6.1. Flow Cytometry Detection of In Vivo Immune System Cell Responses in Tumour
and Spleen
After completing the treatment, the mouse tumours and spleens were aseptically
removed and used to make single-cell suspensions using 70 µm filters (BD Biosciences)67, 68 .
A sharp scalpel was used to cut the tumours and spleens into pieces. The tumour and spleen
tissues were quickly placed in 10 mL of the dissociation buffer (100 U/mL of collagenase IV
and 100 µg/mL DNase I in RPMI media with 10% FBS), then stored at 37 ◦ C in an incubator
for 30 to 40 min. Once the 70 µm strainer was wet after adding 1 mL of cold medium
to a 50 mL falcon tube, the spleens or tumours were placed on it. The organs were then
lightly mashed before being passed through strainers to create single-cell suspensions. The
splenocytes and tumour cells were then collected by washing the strainer with 4 mL of cold
Cancers 2023, 15, 3748 6 of 19

medium. The medium was aspirated off after the cells were centrifuged at 250× g for 5 min
at room temperature. Finally, 1 mL of medium was used to resuspend the cell pellet, the
purified cells were carefully collected, and the resulting suspension was washed once with
FACS buffer. The cells were counted to yield 2 × 106 cells per FACS tube for each sample.

2.6.2. Antibodies and Flow Cytometry

The single spleen and tumours of the treated groups were incubated with antibodies
in staining buffer (0.1 M PBS, 1 per cent FCS, and 0.1 per cent sodium azide) for 30 min on
ice in order to stain the cell surface markers. The cells were stained with CD4 (GK1.5, PE),
CD19 (eBio1D3 (1D3), PE), CD11b (M1/70, PE), and NK1.1 (PK136, FITC). Furthermore,
the cells were incubated with tetramer (SIINFEKL-H2Kb APC) and a CD8a monoclonal
antibody (53–6.7) (BD Biosciences) to determine tetramer+ CD8+ T cells via flow cytometry
assay. The cell percentages were evaluated using FlowJo, and the graphs were made using
GraphPad Prism.

2.6.3. Determination of Antigen-Specific Antibodies in Serum

Using an enzyme-linked immunosorbent assay (ELISA) test, the levels of TNF-α,
INF-γ and OVA-specific antibodies, including two subtypes of IgG, IgG1, and IgG2a, were
measured. On day 32, serum samples were collected and kept at 4 ◦ C. For the OVA-specific
antibody procedure that followed, OVA proteins were applied to 96-well polystyrene
microplates and incubated overnight at 4 ◦ C. After serially diluting the serum samples
twice in blocking buffer, they were added to the washed microplates. The prediluted
horseradish peroxidase-conjugated goat and anti-mouse antibodies IgG, IgG1, or IgG2a
(Abcam, Waltham, MA, USA) were added to the microplates after 2 h incubation at 37 ◦ C.
Tetramethylbenzidine substrate was added after an additional two hours of incubation, and
the chromogenic process was subsequently inhibited by 2MH2 SO4 . Thereafter, a microplate
reader assessed each well’s optical absorbance at 450 nm (Tecan, Männedorf, Switzerland).

2.6.4. Histopathological Studies

Neutral formaldehyde (10 per cent) was used to fix the removed tumours. The samples
were dehydrated using various concentrations of ethanol (30%, 50%, 70%, 90%, and 100%),
after which the tissues were cleaned in benzene and embedded in low melting-point paraf-
fin wax. Thereafter, 5 µm thick sections were mechanically cut into sections and sequentially
placed on clean glass slides. These slides were then stained with Hematoxylin and Eosin
for examination under a light microscope (LEICA DM 3000, Leica, Shanghai, China).

2.6.5. Assessment of the Adjuvanticity of Ppoly

The experiments were conducted in accordance with the appropriate laws and with
the approval of the Animal Experiment Committee of the Institute of Medical Science,
University of Tokyo. Six-week-old female C57BL6/J mice were obtained from CLEA, Japan.
The OVA protein (10 µg) was mixed with Ppoly (35 µg) in 20 µL of PBS. The mice were
then injected with the sample solutions intramuscularly on days 0 and 14. On day 28, the
serum was collected from the immunised mice. OVA-specific antibodies were measured
as mentioned above by using horseradish peroxidase-conjugated anti-mouse IgG, IgG1,
IgG2c, and IgE antibodies (Southern Biotech, Birmingham, AL, USA). Titers of the OVA-
specific antibodies were determined via log-linear interpolation of the serum dilution value
corresponding to the cut-off absorbance (OD450 of 0.2).

2.7. Statistical Analysis

Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software, Inc.,
La Jolla, CA, USA). The data were analysed using one-way ANOVA (Analysis of Variance).
All samples were analysed in triplicate and are presented as mean ± standard deviation
(SD) for n = 3–5. The significance level was calculated by the p-value. Statistically, a
p value < 0.05 was considered significant.
Cancers 2023, 15, 3748 7 of 19

3. Results and Discussion

3.1. Design Rationale and Characterisation of mRNA Nanoparticles
Our specific goal and the basis of our overall hypothesis was to develop an effective
anti-tumour immunotherapy via the local delivery of mRNA loaded on hyper-branched
cyclodextrin nanoparticles. To be considered a successful gene delivery vehicle, the vector
must be able to condense nucleic acids [37]. To confirm the ability of the synthesised
polymer nanoparticles to interact electrostatically with negatively charged mRNA to pro-
duce Ppoly/mRNA complexes, several functional assays, such as electrophoretic mobility
shift assay (EMSA), particle size analysis, zeta potential analysis, and SEM imaging, were
performed [38]. To evaluate the condensation efficiency of Ppoly with mRNA, an elec-
trophoretic mobility shift assay was performed [39]. As can be seen in Figure S1A, even at
the lowest N/P ratio, both EGFP (right) and OVA (left) mRNAs were almost completely
retained in the wells by Ppoly; however, interestingly, the N/P ration 10:1 showed the
highest encapsulating efficiency (EE), about 90% compared to 55% at 1:1 (Figure 2A), which
demonstrated improved complex formation between the polymer and mRNAs through
ionic interactions. Furthermore, as displayed in Figure 2B, when the Ppoly/mRNA weight
ratio reached 10:1, the Ppoly could successfully condense mRNAs into a particle size of
300 nm with a positive potential (about +5 mV). Therefore, the particle size and shape of
the Ppoly/mRNA complex at that ratio were then further examined by SEM and FTIR
(Figure
Cancers 2023, 15, x FOR PEER REVIEW
2C,D). The SEM image demonstrated that the Ppoly/mRNA complexes 8 of 20
were
spherical in shape. Additionally, the 10:1 N/P ratio FTIR spectrum showed that mRNA
complexes were successfully incorporated into the Ppoly (Figure 2D).

2. (A)2.Encapsulation
FigureFigure efficiency
(A) Encapsulation efficiency of
of Ppoly formRNA.
Ppoly for mRNA. (B)(B)
SizeSize of Ppoly/mRNA
of Ppoly/mRNA complexes
complexes at at
different
different N/PN/P ratios
ratios andand zeta-potential of
zeta-potential of DLS.
DLS.(C)(C)SEM
SEMimage
imageof Ppoly/mRNA at an N/P
of Ppoly/mRNA ratio
at an N/Pof ratio of
10:1. (D) The FTIR spectra of PPoly free, mRNA free and Ppoly/mRNA complexes. The results are
10:1. (D) The FTIR spectra of PPoly free, mRNA free and Ppoly/mRNA complexes. The results are
expressed as means ± SD.
expressed as means ± SD.
3.2. Two-Dimensional (2D) and Three-Dimensional (3D) Spheroid Cytotoxicity and Uptake
Analysis
The cytotoxicity effect of different ratios—1:1, 1:5, and 1:10—of Ppoly free and
complexes with mRNA compared to a commercial lipofectamine agent at concentrations of
3, 6, and 12 µL (routine amount used for gene delivery) in free and in complex with mRNA
was assessed in melanoma cancer cell lines (B16–F10). The results illustrated that Ppoly at
Cancers 2023, 15, 3748 8 of 19

3.2. Two-Dimensional (2D) and Three-Dimensional (3D) Spheroid Cytotoxicity and Uptake Analysis
The cytotoxicity effect of different ratios—1:1, 1:5, and 1:10—of Ppoly free and com-
plexes with mRNA compared to a commercial lipofectamine agent at concentrations of 3,
6, and 12 µL (routine amount used for gene delivery) in free and in complex with mRNA
was assessed in melanoma cancer cell lines (B16–F10). The results illustrated that Ppoly
at different ratios did not show cytotoxicity, while lipofectamine at 6 and 12 µL showed
about 32% and 52% toxicity, respectively (Figure S2A–C). These findings underscore the ad-
vantages of utilizing modified cationic polymers, such as Ppoly, in terms of their enhanced
safety and effectiveness. This could potentially overcome existing delivery barriers and
prove beneficial for various in vivo applications. By minimizing cytotoxic effects, Ppoly
offers a more favourable profile for in vivo applications, ensuring the viability and integrity
of targeted cells [34,35]. Based on the characterisation results, formulations with N/P
ratios of 1:1, 1:5, and 1:10 were chosen for the 2D cellular transfection study to compare
the ability of the Ppoly positive charge to deliver the EGFP-mRNA inside the B16–F10
cells to free mRNA and mRNA in complex with the commercial lipofectamine agent using
fluorescence microscopy and flow cytometry. Strong green fluorescence related to EGFP
protein expression was detected in cells treated with mRNA complexes with Ppoly at ratios
of 1:5 and 1:10 compared to lipofectamine (1:3 ratio) and also free mRNA. It should be
emphasised that the intensity of green fluorescence within the cells reflects the capacity of
the cells to uptake our NPs (Figure 3A). In addition, the quantitative uptake of complexes
containing mRNA-EGFP at different N/P ratios was assessed by using a flow cytometer
after incubation for a certain time, and the uptake efficacy was found to be about 77%,
72%, 26%, and 58% for the mRNA in complex with Ppoly 1:10, 1:5, 1:1, and lipofectamine,
respectively (Figure 3B), while the naked mRNA showed a negligible level of EGFP expres-
sion due to the inefficiency of the cellular entry. These findings, which were in line with
publications in the literature [39–42], indicated that Ppoly loaded with mRNA enhanced
transfection efficiency by postponing the release of mRNA from nanoparticles compared to
free mRNA.
Three-dimensional (3D) cell culture has gained popularity due to its ability to simulate
living cells within micro-assembled supports and devices with a 3D structure tailored to the
microarchitecture of a tissue or organ. Therefore, the ability of the designed mRNA NPs to
enter a 3D spheroid structure was also assessed. To evaluate the uptake and transfection of
the mRNA-loaded Ppoly compared to free mRNA, spheroids of B16–F10 cells were created.
The outcomes showed that, after 24 h of incubation with the spheroid cells, free mRNA was
unable to be taken up by 3D cancer cells (Figure 4), whereas EGFP expression in 3D spheroid
cells transfected by Ppoly-mRNA complexes at that time was significantly higher than
lipofectamine and free mRNA (Figure 4). It is interesting to note that Ppoly loaded with
EGFP-mRNA demonstrated high transfection efficiency in 2D monolayer cells (B16–F10),
while these values in 3D spheroids were lower, as demonstrated by other studies on gene
delivery in 3D spheroids, which found similarly low transfection efficiency [43–46]. The
extracellular matrix (ECM) and high-cell-density multicellular barriers in the deep regions
of 3D spheroids are the main barriers preventing gene delivery agents from penetrating the
spheroids’ cells. These cells are known to have extensive cell contact, increase interstitial
pressure simultaneously, and display significant resistance to chemotherapy and radiation
therapy [47]. We noticed this phenomenon in our study and demonstrated that it holds
true for transfection in both two-dimensional and three-dimensional spheroids. The outer
cell layers in 3D spheroids were the only areas of the peripheral cells that were transfected
by Ppoly/mRNA complexes (N/P ratio 10:1), which could explain this phenomenon.
Furthermore, these findings support those of other studies using the in vitro 3D spheroid
model for gene delivery [46,48], which produced clinical findings indicating poor non-
viral gene transfer effectiveness in vivo in tissues with three-dimensional structures or
solid malignancies.
 incubation for a certain time, and the uptake efficacy was found to be about 77%, 72%, 26
and 58% for the mRNA in complex with Ppoly 1:10, 1:5, 1:1, and lipofectamine, respectiv
(Figure 3B), while the naked mRNA showed a negligible level of EGFP expression due
the inefficiency of the cellular entry. These findings, which were in line with publications
Cancers 2023, 15, 3748 the literature [39–42], indicated that Ppoly loaded with mRNA enhanced 9 of transfecti
19

efficiency by postponing the release of mRNA from nanoparticles compared to free mRN

Figure 3. (A)
Figure Fluorescence
3. (A) microscopy
Fluorescence microscopy results
results for thefor
freethe free EGFP-mRNA
EGFP-mRNA and in
and EGFP-mRNA EGFP-mRNA
com-
complexes with Lipofectamine and Ppoly. 1—Free mRNA, 2—Lipofectamine, 3—1:1(mRN
plexes with Lipofectamine and Ppoly. 1—Free mRNA, 2—Lipofectamine, 3—1:1 (mRNA/Ppoly),
/Ppoly), 4—1:5,
4—1:5, 5—1:10.
5—1:10. (B)
(B) Flow Flow cytometry
cytometry results for theresults for the free
free EGFP-mRNA andEGFP-mRNA
EGFP-mRNA inand EGFP-mRNA
complexes
complexes with Lipofectamine and CD-NSs. (* p < 0.05, ** p < 0.01, **** p < 0.0001)
with Lipofectamine and CD-NSs (* p < 0.05, ** p < 0.01, **** p < 0.0001).
 our study and demonstrated that it holds true for transfection in both two-dimensional
and three-dimensional spheroids. The outer cell layers in 3D spheroids were the only
areas of the peripheral cells that were transfected by Ppoly/mRNA complexes (N/P ratio
10:1), which could explain this phenomenon. Furthermore, these findings support those
of other studies using the in vitro 3D spheroid model for gene delivery[46,48], which
Cancers 2023, 15, 3748 produced clinical findings indicating poor non-viral gene transfer effectiveness in vivo10inof 19
tissues with three-dimensional structures or solid malignancies.

Figure4. Microscopic
Figure 4. Microscopic fluorescence
fluorescence images
images of 3Dofspheroids
3D spheroids (B16–F10
(B16–F10 cells), mRNA/Ppoly
cells), mRNA/Ppoly nanocom-
nanocomplexes transfection at N/P ratio 10:1 compared to free mRNA-free, and lipofectamine after
plexes transfection at N/P ratio 10:1 compared to free mRNA-free, and lipofectamine after 24 h
24 h of incubation.
of incubation.

3.3. In Vivo Immunotherapeutic Efficacy of mRNA Nanocomplexes

Next, we studied the in vivo pharmacokinetics and therapeutic efficacy of the
Ppoly/mRNA complexes in a melanoma mouse model. Melanoma cases are rising across
the world [49]. Meanwhile, the death rate from melanoma has decreased due to improve-
ments in the management of systemic diseases with immunotherapy and targeted therapies.
However, survival can present several problems, such as second primary melanomas, an
increased chance of developing other skin cancers, and the long-term effects of melanoma
therapy [50,51]. As a result, there is an urgent need to develop new immunotherapies.
To evaluate the in vivo delivery efficacy of mRNA in complexes with Ppoly, free EGFP-
mRNA (10 µg) and EGFP-mRNA in complex with Ppoly at a ratio of 1:10 were injected
intratumourally into female C57BL6/J melanoma cancer mice. To evaluate the ability
of EGFP-mRNA-loaded Ppoly expression in a melanoma tumour site, the fluorescence
intensity was observed at 2, 4, 6, and 24 h after intratumoural injection through an in vivo
imaging system (Kodak live animal imaging) with an excitation wavelength of 480 nm
and an emission wavelength of 510 nm (Figure 5A). The results showed that the fluores-
cence intensity in the group receiving EGFP-mRNA-Ppoly was significantly increased in a
time-dependent manner compared to free mRNA; a group receiving a saline solution was
considered the control group (Figure 5B). This suggests that Ppoly could be considered
an efficient local delivery system due to its ability to concentrate and deliver the mRNA
to the tumour, which is consistent with the findings of other studies [52–54]. Therefore,
we then assessed the in vivo anti-cancer activity of OVA-mRNA free and in complexes
with Ppoly in an induced melanoma animal tumour model. C57BL/6 mice were treated
four times in four-day intervals once the melanoma tumours were palpable (n = 4 mice).
When the tumours became visible and palpable, the size was measured every other day
using a vernier calliper. Mice treated with PBS and Ppoly-free formulations developed
extensive tumours. In contrast, those treated with OVA-mRNA exhibited significantly
slower and negligible tumour growth. Impressively, the complexes of OVA-mRNA with
Ppoly demonstrated approximately three times more tumour suppression compared to
Cancers 2023, 15, 3748 11 of 19

the untreated group, indicating the potential of OVA-mRNA-Ppoly therapy in preventing

the growth of malignant tumours. Interestingly, there were no significant differences in
average tumour weight between the PBS and free-Ppoly groups (Figure 5C). Interestingly,
there were no differences in average tumour weight between PBS and free-Ppoly, while the
average tumour weight in the Ppoly-OVA-mRNA group was significantly lower than in
the other groups (Figure 5D,E). Notably, the mice in the PBS groups had to be sacrificed
by day 25 due to the rapid expansion and excessive size of the tumours. To understand
the change in tumour tissue morphology in the treated groups, they were further checked
via histopathological examination. The microscopic morphology of the group treated with
PBS exhibited solid sheaths of neoplastic cells with fairly vascular, congested blood vessels,
arranged in irregular cords around a blood vessel, and the nuclei of neoplastic cells were
large, ranging in shape from ovoid to round to irregular polygons with high mitotic indexes
(Figure 5F). In the mRNA treatment group, a proportionate increase in vacuolar degen-
eration and the necrotic areas of the tumour was observed along with the characteristics
of degeneration, which include swelling, vacuolisation, rupture, and fragmentation. In
addition, the cytoplasm of degenerating melanoma cells contained varying amounts of
fine, light brown melanin pigment. Mitotic figures were significantly decreased compared
to the untreated group, as the malignant cell population was lower in this group with a
remarkably small blood supply. In contrast, in the group treated with OVA-mRNA loaded
Ppoly, the parenchyma of the tumour mass primarily consisted of necrotic areas containing
tumour cells with pyknotic, karyorrhectic nuclei, and eosinophilic cytoplasm.

Cellular and Humoral Immune System Responses to mRNA Therapy

The initial activation of adaptive immune activity is necessary for the formation of
efficient and long-lasting anti-tumour immunity [55–57]. So, we assessed the variations
in the immune cell populations of both humoral and cellular immunity using isolated
splenocytes and single-tumour cells to confirm whether the effective anti-tumour effect of
OVA-mRNA-loaded Ppoly was due to an endogenous host immune cell response. For this,
firstly, the harvested tumour and spleen single cells were stained with anti-CD8a (cytotoxic
T cells), CD4 (T- helpers), CD19 (B cells), CD11b (macrophages), and NK1.1 (natural killer).
The results showed that mice given OVA-mRNA-Ppoly complexes had higher percentages
of almost all subtypes of leukocytes in the tumour (Figure 6A) and spleen mononuclear
cells (Figure 6B), including B lymphocytes, macrophages, T-helpers, CTL, and NK cells
compared to the OVA-mRNA-free and control groups, which is consistent with the results
of other studies on the effects of mRNA vaccines designed to stimulate cellular and humoral
immune responses against foreign antigens [58,59].
The mRNA molecules reach the cytoplasm after vaccination, where they are translated
into proteins. Cytosolic proteasomes break down dendritic cells (DC), producing proteins,
and the resultant epitope peptides are then delivered via the MHC class I pathway, which
triggers an antigen-specific CD8+ T cell response [60]. Moreover, the uptake of the extra-
cellular antigens by DCs will induce a CD4+ helper T cell response [61]. Therefore, we
hypothesised that the OVA mRNA is translated into OVA protein, where it is processed by
the immunoproteasome, which is then processed to enable effective antigen presentation
by the antigen-presenting cells (APC) to CD8+ T lymphocytes [57]. Thus, we quantified
the OVA antigen in tumour and spleen cells, which consist of a variety of immune cell
populations [62,63] excised from each group of the treated mice using flow cytometry. Inter-
estingly, mice treated with the OVA-mRNA-Ppoly complex had expressed approximately
11% MHC class I -OVA peptides in tumour cells (Figure S1B) and 4.5% in spleen cells, two
times more than free-OVA. This suggests that the Ppoly can deliver the mRNA efficiently
and that it enhances antigen presentation by MHC class I peptides at the site of tumour cells.
Furthermore, the expansion of OVA-specific CD8+ T cells was evaluated using tetramer+
(SIINFEKL-H2Kb APC) and CD8a for the detection of H-2Kb/SIINFEKL (OVA)-specific
CD8-T+ cells [55]. As shown in Figure S1B,C, compared with untreated mice, OVA-mRNA
complexes with Ppoly resulted in a significant increase in CD8+Tetramer+ T cells in tumour
Cancers 2023, 15, 3748 12 of 19

and spleen mononuclear cells (0.023% vs. 0.8% and 0.010% vs. 0.4%, respectively). The
increased potency of OVAmRNA complexes with Ppoly compared to OVAmRNA alone
was abundantly supported by tumour growth data. The suppression of tumour growth in
the OVA-mRNA-Ppoly-treated group can be attributed to the increased number of OVA-
specific CD8+ T cells. This elevated presence of CD8+ T cells enhances the recognition
of specific tumour cell antigens by cytotoxic T lymphocytes (CTLs). Consequently, the
CTLs release cytotoxic molecules such as perforin and granzymes into the tumour cells,
inducing cellular damage. This process activates caspases, which are enzymes responsible
for initiating the apoptotic cascade, leading to the death of the tumour cells. Furthermore,
previous studies have indicated that this mechanism also promotes the proliferation of
CTLs, contributing to the overall suppression of tumour growth [15,64,65]. Moreover,
serum samples were collected, and the levels of induced OVA-specific total immunoglobu-
lin G (IgG) and IgG subclasses (IgG1 and IgG2a) were measured to confirm the humoral
immune responses to various formulations. According to the results shown in Figure 6C,
OVA-mRNA-Ppoly significantly increased the levels of total IgG, IgG1, and IgG2a com-
pared to naked mRNA. Free Ppoly did not induce humoral responses, confirming that
Ppoly alone cannot induce antigen-specific immune responses. Notably, a naked mRNA
injection could produce moderate humoral immune responses. This effect was observed
previously, and investigations have since demonstrated that various humoral responses
can be partially induced by mRNA injection [55]. These results suggest that Ppoly formula-
tion could indeed enhance the cellular delivery of mRNA and result in stronger humoral
immune responses, which may lead to an increase in antibody-dependent cell-mediated
cytotoxicity. Both successful antigen presentation and appropriate immunostimulatory
signals must be produced for immunotherapy to be effective. Therefore, the serum levels
of proinflammatory cytokines were assessed to confirm the immune-stimulating effect of
OVA-mRNA-Ppoly. The serum levels of the tumour necrosis factor (TNF-α) and interferon
gamma (IFN-γ), which are important markers of a strong immune response, were measured
using an enzyme-linked immunosorbent assay (ELISA). Interestingly, when compared to
the untreated and naked mRNA-treated groups, the OVA-mRNA-Ppoly complex induced
higher IFN-γ and TNF-α levels. This highlighted the synergistic immune-stimulating
effects of Ppoly-encapsulating mRNA (Figure 6C). These findings demonstrate that, in
comparison to mRNA alone, OVA-mRNA-Ppoly could boost cellular immune responses. It
has been shown that TNF-α secretion mainly leads to increased apoptosis and inflamma-
tion, while IFN-γ secretion leads to an increase in CTL cell activity and Th1 differentiation,
which in turn can activate macrophages in a classical pathway to increase inflammation in
targeted cells [66]. Therefore, both the strong stimulation of cellular and humoral immune
system responses and high-level secretion of these cytokines could partially explain the
antitumor effect of OV-mRNA loaded on Ppoly. To further verify the robust immunos-
timulatory effect mechanism of the mRNA–Ppoly complex, we examined whether Ppoly
possesses adjuvanticity by intramuscularly immunising the normal mice with a mixture of
the OVA protein and Ppoly. As a result, the production of OVA-specific IgG1 was increased
in the presence of Ppoly, although there was no change in that of OVA-specific IgG2c
(Figure 7A). The levels of antigen-specific IgG1 and IgG2c reflect the induction of Th2 and
Th1 responses, respectively [67]. Therefore, these data suggest that Ppoly can moderately
increase the antigenicity of the loaded OVA protein compared to free OVA via an increased
induction of Th2-related immune responses.
Cancers 2023,
Cancers 15, 3748
2023, 15, x FOR PEER REVIEW 1213of
of 20
19

Figure 5. (A)
Figure 5. (A) Typical
Typical in vivo images
in vivo images of
of female
female C57BL6/J
C57BL6/J mice,
mice, inratumoural
inratumoural injection
injection of
of saline
saline
(control group),free
(control group), freemRNA,
mRNA,and and mRNA
mRNA in complex
in complex withwith Ppoly
Ppoly all atall at a dosage
a dosage of EGFP-mRNA.
of 10 µg 10 µg EGFP-
mRNA. (B) Quantitative
(B) Quantitative assay of fluorescence
assay of fluorescence intensity intensity from theof
from the tumours tumours
differentofgroups
different groups
based on inbased
vivo
on in vivo images. (C) The anti-tumour growth effect of OVA-mRNA both free and in complexes
images. (C) The anti-tumour growth effect of OVA-mRNA both free and in complexes with positively
with positively charged Ppoly. (D) The mice of the PBS, OVA-mRNA, free Ppoly, and OVA-mRNA
charged Ppoly. (D) The mice of the PBS, OVA-mRNA, free Ppoly, and OVA-mRNA + Ppoly groups
+ Ppoly groups and their excised tumours were imaged at day 25 and 32 post-tumour induction,
respectively. (E) Tumour measurements were performed daily using callipers, and the average
tumour volume was calculated as length × width2 × 0.52 to represent the average of tumour weight
Cancers 2023, 15, 3748 14 of 19

and their excised tumours were imaged at day 25 and 32 post-tumour induction, respectively.
(E) Tumour measurements were performed daily using callipers, and the average tumour volume
was calculated as length × width2 × 0.52 to represent the average of tumour weight and statistical
size differences among the groups. (Error bars represent the SD, and significance was determined
using One-Way ANOVA (ns, Not Significant; * p < 0.05, *** p < 0.001, # p < 0.0001)). (F) The microscopic
appearance of tumour mass from the group treated with “PBS”. The tumour cells were arranged
in nests or as cords around the blood vessels and mitotic figures (arrows) exhibiting a remarkably
small blood supply with the invasion of tumour cells into the blood vessels (arrows) and mitotic
figures (arrow heads). The OVA-mRNA-treated group indicated extensive tumour cell vacuolation
(arrows), swelling, rupture, and fragmentation, as well as a prominent decrease in cell population; the
neoplastic tissue was rather degenerated, and characteristics of degeneration were more prominent
Cancers 2023, 15, x FOR PEER REVIEW 15 of 20
with the decrease in cell population. Furthermore, the OVA-mRNA +Ppoly-treated group’s tumour
tissues were rather necrotic, with characteristics of coagulative necrosis with the massive necrosis of
melanoma cells (H&E, 400×).

Figure 6. The cellular immune system response in C57BL/6 mice tumour and spleen tissues. On the
Figure 6. The cellular immune system response in C57BL/6 mice tumour and spleen tissues. On the
final day of the treatment period, (A) the tumours and (B) the spleens were excised and homogenised,
final day of the treatment period, (A) the tumours and (B) the spleens were excised and
and immune celland
homogenised, infiltration
immunewas cellanalysed via flow
infiltration was cytometry.
analysed via Antigen-specific
flow cytometry. antibodies induced
Antigen-specific
antibodies
by OVA-mRNA induced
+Ppolyby nanocomplexes.
OVA-mRNA +Ppoly nanocomplexes.
(C) After the end of the(C) After the
treatment end the
period, of the treatment
OVA-specific
period, theincluding
antibodies, OVA-specific
serum antibodies, including
IgG antibodies, IgG1serum IgG antibodies,
antibodies, and IgG2aIgG1 antibodies,
antibodies, were and IgG2a
examined
antibodies,
in the serum.were examinedthe
In addition, in serum
the serum.
level In addition,
of TNF-α andthe serum
IFN-γ waslevel of TNF-α
assessed and IFN-γ
in different was
groups.
assessed
(Error barsinrepresent
different groups. (Error
the SD, and bars represent
significance was the SD, and significance
determined using One-Way was ANOVA
determined(ns,using
Not
One-Way ANOVA
Significant; (ns,
* p < 0.05, ** Not Significant;
p < 0.01, * p < 0.05,
*** p < 0.001, p < 0.01, *** p < 0.001, # p < 0.0001).
# p <**0.0001).
 Cancers
Cancers 2023,2023, 15, x FOR PEER REVIEW
15, 3748 16 of
1520
of 19

Figure7. 7.(A)
Figure (A)The
Thehumoral
humoral immune
immune system responseofofimmunised
system response immunisedmicemice treated
treated with
with free
free OVAOVA
protein(10
protein (10µg)
µg)and
andOVA
OVAprotein
protein in
in complexes
complexes with
with Ppoly
Ppoly(35
(35µg)
µg)bybyevaluating
evaluating OVA-specific
OVA-specific
antibodies,including
antibodies, includingserum
serum IgG
IgG antibodies,
antibodies, IgG1
IgG1antibodies,
antibodies,and
andIgG2a
IgG2a antibodies,
antibodies,in the plasma.
in the plasma.
(B) The IgE production by immunised mice treated with free OVA protein (10 µg) and the OVA
(B) The IgE production by immunised mice treated with free OVA protein (10 µg) and the OVA
protein in complexes with Ppoly (35 µg). (ns, Not Significant; * p < 0.05, ** p < 0.01, *** p < 0.001).
protein in complexes with Ppoly (35 µg). (ns, Not Significant; * p < 0.05, ** p < 0.01, *** p < 0.001).
4. Conclusions
Given that many vaccinations are intended to be administered to healthy individuals,
one of The foundations
the most crucial were laid of
qualities forvaccine
the rapid development
adjuvants, of mRNA
in addition vaccinations
to their during
efficiency, is their
the COVID-19 pandemic by years of research into mRNA vaccines for cancer treatment
safety. Traditionally, it has been considered that Th2 and IgE (an allergic antibody) responses in
preclinical
are sequential and clinical
[68]; trials. we
therefore, Thechecked
inherentthe
benefit of ease of levels
IgE secretion production,
in mice which rivals
treated thethe
with
bestprotein
OVA conventional
(both vaccine
free and manufacturing
in complexesmethods currently
with Ppoly) available,
because makesoftherapeutic
the ability an adjuvant
tocancer
inducevaccines
IgE may based
be aon mRNA
crucial an factor
risk attractive optionthe
affecting for allergenic
cancer immunotherapy. These
potential of vaccines.
vaccines are well-tolerated and have the potential to be a powerful tool
The data suggested that Th2 induction does not always induce strong IgE production in the fight against
cancer [71].
because Ppoly at defined concentrations did not lead to the production of IgE in mice
(FigureIn7B).
this study, we conducted a comprehensive evaluation of the efficacy and safety of
This result was consistent with the findings of a study by Hayashi et al., which
Ppoly as an mRNA delivery vehicle. Initially, we demonstrated that Ppoly efficiently
demonstrated the effective adjuvant ability of Hydroxypropyl-β-Cyclodextrin complexes
delivered EGFP-mRNA to both 2D and 3D melanoma cell lines, surpassing the delivery
with the OVA protein [69,70]. As a result, these data suggest that Ppoly in complexes with
efficiency of naked mRNA. Importantly, we observed no cytotoxic effects associated with
the OVA protein could induce Th2-related immune responses in a safety pathway, while
Ppoly, further establishing its safety as a delivery system. These findings underscored the
the mRNA–Ppoly complex induced not only Th2 but also Th1 responses, such as increased
potential of Ppoly as a robust and secure mRNA delivery vehicle, as confirmed by in vitro
CD8+ T cells, antigen-specific IgG2a, and IFN-γ production (Figure 6A–C). Collectively,
cytotoxicity assays.
complexation between therapeutical mRNA and Ppoly may lead to the robust induction of
Building upon these promising results, we proceeded to evaluate the biodistribution
cellular immune responses
of mRNA-EGFP followingincluding the Th1,
intratumoural Th2, and
injection in CTLs, whichinduced
mice with are mainly expected
melanoma
tocancer.
contribute to immunotherapeutic effects in the tumour model.
The time-dependent analysis revealed that EGFP expression was higher and more
sustained over time when delivered in complexes with Ppoly compared to free EGFP.
4. Conclusions
The foundations were laid for the rapid development of mRNA vaccinations during
the COVID-19 pandemic by years of research into mRNA vaccines for cancer treatment in
Cancers 2023, 15, 3748 16 of 19

preclinical and clinical trials. The inherent benefit of ease of production, which rivals the
best conventional vaccine manufacturing methods currently available, makes therapeutic
cancer vaccines based on mRNA an attractive option for cancer immunotherapy. These
vaccines are well-tolerated and have the potential to be a powerful tool in the fight against
cancer [71].
In this study, we conducted a comprehensive evaluation of the efficacy and safety
of Ppoly as an mRNA delivery vehicle. Initially, we demonstrated that Ppoly efficiently
delivered EGFP-mRNA to both 2D and 3D melanoma cell lines, surpassing the delivery
efficiency of naked mRNA. Importantly, we observed no cytotoxic effects associated with
Ppoly, further establishing its safety as a delivery system. These findings underscored the
potential of Ppoly as a robust and secure mRNA delivery vehicle, as confirmed by in vitro
cytotoxicity assays.
Building upon these promising results, we proceeded to evaluate the biodistribution
of mRNA-EGFP following intratumoural injection in mice with induced melanoma cancer.
The time-dependent analysis revealed that EGFP expression was higher and more sustained
over time when delivered in complexes with Ppoly compared to free EGFP.
Next, we investigated the anti-tumour effects of intratumourally injected OVA-mRNA
loaded on Ppoly. The results showed a significant decrease in both tumour size and weight
in the group treated with OVA-mRNA in complexes with Ppoly compared to other formu-
lations. Furthermore, we sought to understand the immunological mechanisms underlying
the observed anti-tumour effects. We discovered that OVA-mRNA, as a foreign antigen,
complexed with Ppoly, elicited a robust adaptive immune response in vivo. This was
evidenced by the substantial increase in most leukocyte subtypes and the remarkable
expansion of OVA-specific CD8+ T cells observed in both spleen and tumour tissues, sur-
passing the immune responses observed in untreated groups. Additionally, OVA-mRNA,
in combination with Pploy, significantly improved humoral immune responses in mice
with induced melanoma tumours. These facts were considered in the new immunotherapy
strategy, which resulted in efficient anti-tumour immunity against melanoma cancer and
significantly inhibited tumour growth. The host immune system detects tumour cells that
display non-self-foreign antigens as foreign or infected cells [54], which is the reason why
the delivery of foreign antigens via polymeric complexes can have anti-tumour effects. The
results of this work demonstrate that the intra-tumoural administration of a cationic hyper-
branch cyclodextrin-based polymer containing a foreign antigen—OVA-mRNA—may be a
new and promising therapy to promote the immunological treatment of melanoma cancer,
with a moderately adjuvant ability to induce Th2-related immune responses in a safety
pathway. However, more in-depth studies are required to better understand the delivery
mechanism and enhance the efficiency of the mRNA inside this polymer; comparisons
between this system and other existing carriers are also required.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/cancers15143748/s1. Figure S1. (A) Gel retardation assay for OVA,
left, and EGFP-mRNA, right. (B) Quantification of H2-Kb SIINFEKL expression and SIINFEKL+
CD8+ T cells antigen specific T cell responses in tumor tissues, and (C) Spleen tissues following
treatment with formulations. Figure S2. (A) Cytotoxicity results for different concentrations of Ppoly.
(B) Different ratios (1:1, 1:5 and 1:10) of Ppoly free and complexes with mRNA. (C) Different volumes
of Lipofectamine (µL) free and complexes with mRNA against melanoma cancer cell lines.
Author Contributions: Conceptualisation, Y.K.M., P.Z.-M. and F.T.; data curation, Y.K.M., M.M.,
P.Z.-M., A.M. and F.T.; funding acquisition, F.T.; investigation, Y.K.M., F.T. and M.M.; methodology,
Y.K.M., M.M. and T.H.; project administration, Y.K.M. and F.T.; validation, Y.K.M., M.M., C.C., P.Z.-M.
and F.T.; visualisation, Y.K.M.; writing—original draft, Y.K.M.; writing—review and editing, M.M.,
F.T., P.Z.-M., A.M., J.C., T.H., K.J.I., C.C. and F.T. All authors have read and agreed to the published
version of the manuscript.
Cancers 2023, 15, 3748 17 of 19

Funding: This research was partially funded by The Italian Ministry of Enterprises and Made in
Italy (project acronym CN-RNA) under the PNRR among the initiatives aimed towards creating an
integrated system of research and innovation infrastructures (PNRR M4C2 PROJECTS).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board (Ethics Committee) of Tabriz University of Medical Sciences, Pharmacy department,
Iran (Ethical Code: IR.TBZMED.AEC.1402.001).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article (and
Supplementary Materials).
Acknowledgments: This work is the result of a contract for the University of Turin (Italy) for Training
(For Y.K.M.) and for A.M. and a RTDA contract from the D.M 1062/2021 (Ministero dell’Università e
della Ricerca) for the University of Turin. This research acknowledges support from the Project CH4.0
under the MIUR program “Dipartimenti di Eccellenza 2023–2027”. J.C. acknowledges that they have
a contract with the European Research Council—ERC Starting Grant 848325 for financial support.
Conflicts of Interest: J.C. is a co-founder and shareholder of TargTex S.A. The other authors declare
no conflict of interest.

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