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172 J. Li et al.
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the weak-immunostimulatory PSiNPs-based core, the nanovaccine was still able to
elicit the DCs activation and the following antitumor immune responses (Fig. 8.2j,
). The results of the in vivo experiments further indicated that the injected
k
nanovaccine can
boost the anticancer immune response to prevent solid tumor
occurrence and finally can prolong the mice survival time. In addition, with the
combination of the photothermal effect of the PSiNPs@Au nanocore and the
assistance of the anti-CTLA-4 antibody, the photothermal therapy-induced
antitumor immune response and the following TME reversion endowed the
nanovaccine capac
it
y to significantly
inhi
the 4 T1 solid tumor growth and
bit
suppress metastatic tumor occurrence.
The immunoregulatory effects of the PSiNPs are different and predominately
based on the surface chemistry. In addition to the use of these systems for cancer
immunotherapy, Flavia et al. also developed two potential therapeutic agent-delivery
nanoplatforms based on the PSiNPs for the treatment of autoimmune diseases
represented by Rheumatoid arthritis (Fontana et al.
2018). The UnTHCPSi NPs
and TCPSi NPs were separately coextruded with the KG-1 macrophages membrane
to form the UnTHCPSi@KG-1 and TCPSi@KG-1 nanoplatforms. The in vitro
immunological profile results indicated that after the co-incubation with KG-1
macrophages, the UnTHCPSi@KG-1 or TCPSi@KG-1 both did not significantly
activate the expression of the CD80 and CD86. Moreover, with the KG-1 macrophage membrane coating, the immunostimulatory effect of the UnTHCPSi was
attenuated (Fig. 8.2l).
8.3 Porous Silica NPs
In addition to PSiNPs, another major porous Si-based nanomaterial is the porous
silica NPs. The use of silicon dioxide (SiO
known, has gained much attention in recent years regarding their use in immunotherapy. The reported studies have shown that this type of material has the ability to
act as an adjuvant in immunotherapy, as it can improve the cell uptake capacity of
agents (Ding et al. 2018; Wang et al. 2016). Furthermore, its biocompatibility,
multifunctionality, and tunable properties make it an attractive nanocarrier for the
delivery of immunogenic materials (Carvalho et al. 2020; Chen and Cong 2023; Yu
et al. 2022). Although there are several ways to synthesize these types of NPs, the
most commonly used is the Stöber method, a sol-gel process in aqueous solution.
This process consists of the formation (at low temperature) of an inorganic polymeric network, having as a product a gel from a colloidal suspension or by hydrolysis followed by condensation of the silica precursor, also in solution (Grün et al.
1997; Stöber et al. 1968
).
) NPs, or silica NPs as they are also
2
8 Porous Si-Based Nanosystems for Immunotherapy Applications 173
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8.3.1 Porous Silica NP-Based Immunotherapy
In this section, we will discuss some typical porous silica NP-based immunotherapy
research as examples to introduce the construction of the nanomaterials and their
specific applications for immunotherapy.
In addition to the advantages mentioned above, SiO
of being easily functionalized. Benjamin et al., compared different functionalization
moieties (with amino and carboxy groups) in order to assess whether the different
levels of the functional groups would interfere with the loading of birch pollen
allergen Bet v 1 for allergen-specific immunotherapy, since different binding capacities could influence biological absorption, which would contribute to the overall
safety profile of the system (Punz et al.
2022). First, the SiO
by the Stöber method and then further functionalized. Before binding with Bet v
1, the bare and both functionalized NPs were evaluated in order to exclude possible
endotoxin contamination by the monocyte activation test and HEK Blue
detection assay, since its presence, in addition to compromising the quality of the
final product, could also compromise the in vitro tests. Regarding the Bet v1
coupling, a percentage of 21.75, 11.41, and 0.1% was observed for SiO
without functionalization, functionalized with -COOH and -NH
thermore, it was observed that functionalization did not affect the viability of the
APCs, in this case DCs, which were chosen because they lead to the initiation of a
primary immune response. Additionally, nonsignificant differences in the maturation
status of the DCs were induced by the differently functionalized SiO
et al. conclu d ed that this characteristic of being an inert carrier made this type of NP
ideal for carrying immunological modifiers.
Ovalbumin, a protein from chicken eggs, is known to be a model antigen used in
the development of immunotherapies (Pang et al. 2022; Hou et al. 2023; Xu et al.
2022a). In another study, aiming at an antitumor activity, Lee et al. conjugated
ovalbumin onto silica-coated magnetic NPs (MNPs@SiO
related literature on the association of antigens with NPs to increase the function of
DCs motivated the authors to carry out this work. However, due to the problems of
toxicity inherent in some types of NPs, this made SiO
coating a cobalt ferrite core. In order to verify the final system morphology, and
confirm that they were properly formed, ovalbumin labeled with fluorescein isothiocyanate and rhodamine isothiocyanate dye-bonded silica were used. In addition, it
was observed that the developed system was able to not only increase the T helper
cells 1 and cytotoxic T lymphocyte-mediated immune response but also to exhibit
antitumor activity without toxicity, proving that the system was an interesting
system for the treatment of cancers (or other immune disorders) with potentially
other antigen options.
Bevacizumab is one of the most used monoclonal antibodies for the treatment of
several types of cancer due to its antiangiogenic activity, however, it is also widely
used for the immunotherapy of chronic diseases such as macular degeneration.
Nevertheless, when the treatment strategy consists of crossing some barrier, such
NPs also have the versatility
2
NPs were synthesized
2
TM
2
, respectively. Fur-
2
NPs. Benjamin
2
) (Lee et al. 2019). The
2
NPs the one of choice for
2
LPS
NPs
174 J. Li et al.
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as the blood–brain barrier or the blood–retinal barrier, strategies must be employed
in order to obtain the expected therapeutic result (Sousa et al. 2018, 2019; Andrew
et al. 2010). The major challenge involved in crossing the blood–retinal barrier is the
short drug half-life in the vitreous, which leads to the application of several doses
(by intravitreal injection) with high concentrations (Andrew et al.
2010; Schulz and
Szurman 2022). In order to circumvent these limitations in the treatment of macular
degeneration, which is a chronic disease, Andrew et al. developed nanostructured
films using the technique of electrochemical attack and thermal oxidation in
PSiO
2
air (at 800 °C) (Andrew et al. 2010). From the results of the enzyme-linked
immunosorbent
assay, Andrew et al. observed a loading of approximately 250 μL,
and approximately 94% of the drug was released in a period of 1 month. This was an
interesting finding, since for this disease to be kept under contr ol, the antiangiogenic
drug must be used for a longer period.
Due to the unfavorable factors, for example, the spontaneous urination or the
degradation of the chemodrugs, it is challenging to treat bladder cancer (Dalton et al.
1991; Benson et al. 1980; Maeda et al. 2011). To overcome the obstacles, Chen et al.
fabricated hollow silica NPs functionalized with thiol groups on their external
surface to form a thiolated nanovector for the bladder cancer treatment (Chen et al.
2023). Besides the enhanced mucoadhesion effect, the fabricated nanoplatform can
also stimulate the polarization of macrophages. Moreover, the hollow structure could
endow the nanovector with a large capacity to load the mitomycin C as
the chemotherapeutic agent. In addition to confer a mucoadhesive characteristic to
the system, which was confirmed by ex vivo experiments with a porcine bladder, the
thiol groups provided a better permeation capacity to the formulation, by opening the
tight junction, which consequently increased the delivery of the chemotherapeutic
drug to its target site. Furthermore, in both in vitro and in vivo assays, compared with
the therapeutic outcomes obtained with the free chemotherapy drug-treated group,
those treated with the fabricated nanovector group showed better antitumor activity
and induced macrophage reprogramming, from the M2-phenotype to M1-like phenotype. With those results, the fabricated nanovector was considered as a novel
option for bladder cancer treatment.
It has been reported that Glycoalkaloids (GA) can be used for cancer treatment
(Nepal et al.
2019), but their hemolytic properties hinder the therapeutic applications
(de Groot and Müller-Goymann 2016). To avoid this problem, Nepal et al. developed an amino-functionalized mesoporous silica NP (MSN)-based nanosystem to
load α-tomatine, a GA from tomato, against cancer cells (Nepal et al.
2023
). The
fabricated nanosystem was coated with biodegradable polydopamine (PD) and
polyethylene glycol (PEG). The characterization results showed that the coating
can endow the nanosystem with the capacity to respond to pH changes, reduce the
possible phagocytosis, and prolong blood circulation. Particularly from the drug
release results, it can be seen that at pH = 5.5, approximately 80% of the drug was
released in 24 h, while at pH = 7.4, only 25% was released in the same period of
time. In addition, compared with the hemolysis result in the free α-tomatine group,
due to the PD and PEG coating, the hemolysis in the nanosystem group was
decreased significantly, with almost no sign of hemolytic activity. The in vitro
8 Porous Si-Based Nanosystems for Immunotherapy Applications 175
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experiments carried out in human liver cancer cells and normal liver cells showed
that a comparable level of toxicity was observed between the nanosystem group and
the free α-tomatine group. In normal cells, the nanosystem showed less toxicity at
intermediate concentrations (1.6 and 3.2 μg/mL). Furthermore, it was also observed
that the cancerous cells showed signs of late apoptosis and the cell death mechanism
was caspase-mediated apoptosis, as defined by the caspase
inhibition assay.
As mentioned in the last section, nanovaccine can be used for cancer immunotherapy, but the immunos uppressive TME hinders the anticancer nanovaccine
(Meng et al.
nanovaccine, Zhao et al. developed a nanovaccine based on the SiO
2022; Wang et al. 2022). To improve the therapeutic effect of the
NPs loaded
2
with R837 (used as an immunological adjuvant) coated with hybrid membranes from
cancer cells and DCs via the coextrusion (Zhao et al. 2022). The NPs without coating
showed an encapsulation efficiency of 85.7% while the coated one showed a value of
89.1%. The release experiments were carried out in pH = 5 and pH = 7.4 conditions
and the uncoated NPs showed much higher values than the coated NPs at both pH
conditions. However, values from the coated NPs were much higher than those of
uncoated NPs on the 8th day. However, it should be noted that on the first day of
release, the minimum value observed was 42% drug release. Thus, from in vitro
experimental results, it was observed that the coated NPs proved to be a durable
source of the loaded immunoa djuvant and the hybrid membrane-coated system can
promote endocytosis of the antigen in DCs. Finally, comparing both NPs, Zhao et al.
observed that porous silica-based nanovaccines have a greater capacity to stimulate
the CD8 T cells-related immune response for tumor growth inhibition, and the
nanovaccine also can prolong the survival time of tumor-bearing mice, modulate
the TME, and suppress the recurrence of the tumor.
Some commonly used vaccines c urrently have inactivated or attenuated pathogens as antigens. In order to prevent the rapid degradation of those antigens, Hou
et al. decided to use nanoparticles as carriers. It is worth mentioning that in this case,
the SiO
NPs were synthesized with a topological structure, similar to antigens
2
(flower-like) (Fig. 8.3) since their rough surface would bind more strongly to the
organism’s membranes (Hou et al. 2022
). For virus-like particles (VLPs), the
encapsulation efficiencies of three mass ratios, 1:6, 1:3, and 1:12 (VLPs:
nanoparticles) were quantified by western blotting (results were 92.0%, 83.4%,
and 92.0% encapsulation efficiency, respectively). Of these, Hou et al. chose the
1:6 ratio to continue the study. The release profile of the system was also analyzed
using the Western blotting technique and it was observed that 50% of the VLPs were
released in 24 h, followed by a sustained release of 60% up to 72 h. Additionally it
was observed that the nanoparticles not only increased the stability of the VPLs but
also improved the cellular uptake efficiency. When comparing the free and incorporated VPLs in the in vivo tests, it was observed that the latter induces higher levels of
specific antibodies, greater activation of lymphocytes, and estimated greater secretion of cytokines. Furthermore, as the route of administration was nasal, it was also
observed that the system promoted an immune response in the mucosa of the
animals. It was concluded that these extremely promising results lead this system
to be considered useful for the development of other vaccines.
176 J. Li et al.
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Fig. 8.3 Morphology of the flower-like porous silica NPs. (a) and (b) TEM images of the NPs. (c)
Scanning electron microscope images of the NPs. (Copyright © 2022, Elsevier)
Besides, to avoid the pain during the vaccine injection, Guo et al. synthesized
microneedles (MNs) as a painless vaccine for the tumor immunotherapy, the developed microneedle was mainly composed by biocompatible, biodegradable, and
nontoxic chitosan containing the tumor antigen, OVA257 – 264 fused with hepatitis
B core protein virus-like particles (OVA-HBc) and silica NPs, as vaccine adjuvant
(Guo et al. 2021). The results of the OVA -HBc release showed that with the silica
NPs inside the MNs, after 8 h, much more antigen, OVA-HBc were released when
compared with the chitosan-MNs without silica NPs. The in vivo immunological
experiments further indicate that the chitosan/silica NP-based MNs can induce the
highest amount of the T cells in the inguinal lymph node compared with other groups
(Fig.
8.4a–d). Besides, the in vivo tumor-prevention (Fig. 8.4e–g) and antitumor
experiments indicate that the chitosan/silica NP-based MNs can induce the antigenspecific antitumor immune response and effectively inhibit the tumors’ growth.
8.4 Conclusions
In this chapter, we summarized and discussed the typical porous Si-based
nanosystems for immunotherapy, mainly PSiNPs-based immunotherapy
nanosystems and porous silica-based nanosystems. With the advantages of the
PSiNPs and the porous silica NPs, such as the small size, high drug-loading capacity
or the inherent immunoregulatory effect, these immunostimulatory or immunosuppressive nanosystems can more effectively induce the specific immune responses for
cancer immunotherapy. In addition, the excellent loading and delivery capacity of
the porous Si NPs increase the availability and effectiveness of the simultaneous
delivery of multitherapeutic agents to the targeted immune cells, which can further
enlarge the therapeutic efficacy and persistence of the immunotherapy. Moreover,
the excellent in vitro and in vivo biodegradability and biocompatibility of the
porous-Si NPs endow the nanosystems, promising clinical translation to immunotherapeutic applications.
8 Porous Si-Based Nanosystems for Immunotherapy Applications 177
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Fig. 8.4 In vivo immunological evaluation and tumor-prevention ability evaluation of the chitosan/
silica-based microneedles.
Percentage of CD4- and CD8-positive cells in CD3-positive cells in inguinal lymph node. (d)
Statistics data of CD8-positive cells in CD3-positive cells. (e) The scheme of the vaccination
timeline and the tumor challenging. (f) The in vivo bioluminescence images of the mice after the
tumor inoculation. (g) Tumor growth curve after the tumor inoculation. (Copyright © 2023,
Elsevier B.V)
(a) The scheme of the vaccination. (b) Vaccination site circled in red. (c)
However, several limitations still exist in the current porous-Si immunotherapy
nanosystems, which need to be investigated further, such as a lack of targeting ability
to the aimed immune cells, the possible leakage of the nanosystems into the blood,
accurate injected dosage, and a very important one, how to avoid systematic
inflammation or cytotoxicity.
Acknowledgments Prof. H.A. Santos acknowledges financial support from UMCG Research
Funds and the Academy of Finland (Grant 331151). J. Li acknowledges financial support from
CSC scholarship. G. C. Carvalho acknowledges the financial support from São Paulo Research
Foundation (FAPESP, Brazil) grant number n°.22/02187-0.
178 J. Li et al.
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