Review

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REVIEW
<<Please check the yellow highlights>>BM|d2bm00011c|10.1039/d2bm00011c|REV
Engineering optimal vaccination strategies: effects of physical properties of the delivery
system on functions
Ge Zhu,a,b Yong-Guang Yanga,b,c and Tianmeng Sun*a,b,c
a
Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, Institute of Immunology,
The First Hospital, Jilin University, Changchun, Jilin, China. E-mail: tsun41@jlu.edu.cn
b
National-local Joint Engineering Laboratory of Animal Models for Human Diseases, Changchun, Jilin, China
c
International Center of Future Science, Jilin University, Changchun, Jilin, China
Received 5th January 2022, Accepted 23rd January 2022
This review summarizes the recent advances and insights into the applications of drug
delivery systems (DDSs) in vaccination and the impacts of the major physical properties of
DDSs on the success of vaccines.
GA1
With rapid developments in medical science and technology, vaccinations have has become the key to solving
public health problems. Various diseases can be prevented by vaccinations, which mimic a disease by using a
small part of the causal organism—also called an antigen—to stimulate the specific immune response to defend
the host against the disease. The interaction between antigens and antigen-presenting cells (APCs) affects the
efficacy of a vaccine. Adjuvants are crucial components of vaccinesvaccinations, optimizing the process of
antigen phagocytosis by the APCs to some extent. However, many vaccinations fail during application in
clinical trials owing to drawbacks of some antigens and adjuvants, such as immunogenicity and high
degradability. Owing to rapid developments in the payload, targeted delivery and controllable release of drug
delivery systems, they may have the potential to improve vaccinesvaccinations. In our review, we discuss

Biomaterials Science REVIEW
multiple delivery systems and their role in vaccines and discuss how the major physical properties of delivery
systems ultimately affect the success of vaccines, such as size, morphology, surface charge and elasticity.
1. Introduction
Vaccines Vaccinations are an indispensable component of public health systems, stimulating the immune
system to produce specific immune responses and create protection against the infection and/or disease. Many
diseases, including smallpox, measles, and polio, have been successfully eradicated or have a greatly reduced
incidence owing to the use of vaccines.1. Despite this great progress, vaccination efficacy has been limited by
antigen variability and low immunogenicity. The introduction of adjuvants has largely resolved the problem of
poor antigen immunogenicity and reduced the amount of antigen required, and thus the side effects of
vaccinesvaccinations. However, poor stability, high degradability and unacceptable tolerability of some antigens
and adjuvants limit the efficiency of vaccine application, along with antigen phagocytosis by antigen-presenting
cells (APCs), despite their uptake by APCs triggering the subsequent presentation of antigens and thus
representing an important factor affecting the vaccination efficacy. In 1995, one of the first nanomedicine
doxorubicin liposomal medication, Doxil, was approved for Kaposi’s sarcoma; since then, many material-
composite drugs have been approved by the US Food and Drug Administration (FDA).2–4. Drug delivery
systems (DDSs) have realized degradable biological macromolecular drugs payload, targeted delivery,
controllable release, and favourable pharmacokinetics of drugs.5–9. Therefore, DDSs have the ability to improve
vaccinations. In this review, we illustrate the role and classification of current vaccine delivery systems and
discuss the impact of changes in material properties on the effects of the vaccine, so as to develop more
optimized and suitable vaccine delivery systems in the future.
2. Vaccination mechanisms
Antigens, also called immunogens, are molecules that react with components of the immune system and activate
adaptive immunity. The majority of clinical vaccinations imitate the causative agent of a disease as antigens to
stimulate specific immune responses (Figure Fig. 1). Vaccines can be categorized as live (weakened or
inactivated) and non-live vaccines based on the antigens used. The common types of vaccines that are currently
used are summarized in Table 1. Live vaccines can induce a strong immune response, but there is the potential
for uncontrolled responses in immunodeficient individuals.10. The antigen components of non-live vaccines
include proteins, peptides and polysaccharides.11. Subunit vaccines have a pure composition and high safety, but
This journal is © The Royal Society of Chemistry 2022Biomater. Sci., 2022 | 2

relatively low immunogenicity. Particularly, when faced with the novel coronavirus COVID-19, DNA/RNA
vaccines have great potential for long lasting immune stimulation and anti-pathogen mutation. Inducing
powerful immune responses and maintaining long-term immune memory with low doses of antigen is are a
primary goals of research on contemporary vaccines.
Adjuvants, which are essential for vaccines, are components that enhance and shape antigen-specific
immune responses. Understanding the mechanism of action of different adjuvants is the foundation to
predict the effect of vaccines. Currently, adjuvants can be broadly divided into three categories. The
first comprises immunomodulatory molecules, including pattern recognition receptor (PRR) agonists,
among which toll-like receptor (TLR) agonists are the most widely studied and applied; polycytidylic
acid (poly-IC), an agonist of TLR3, and poly-IC derivatives have been applied in a clinical trial for
tumor treatment,12, and shows potential as an adjuvants in cancer vaccines. Unmethylated 5′-C-
phosphate-G-3′ oligodeoxynucleotide (CpG ODN), an agonist of TLR9, has also been certified as an
adjuvant of vaccines for cancers and infectious diseases in clinical trials.13,14. Another group of
immunomodulatory molecules already used in clinical application are lipid A analogues, such as
monophosphoryl lipid A (MPLA).15. The second category comprises particulate formulations, including
aluminum salts, MF59 and virosomes, which are also used in many clinical vaccines.16–19. This group of
adjuvants also serve as delivery agents. The third category is combination adjuvant systems, in which
two types of adjuvants are combined, eliciting an improved effect. Adjuvant system 01 (AS01,
comprising MPLA and saponin) and AS04 (MPLA and aluminum salt) have been clinically approved,
and have satisfactory efficacy.20,21.
2.1. Injection site reactions
Most vaccines are administered subcutaneously, intradermally, intramuscularly or viavia the mucosal
route,22, and in the clinic, the most commonly used injection method is intramuscular.23. This approach
is superior to intravenous injection, in which the vaccine may be cleared by phagocytes in the
circulation, liver and spleen; intravenous injection is used in only a few cancer therapeutic vaccines.24.
Although the immune response to vaccines predominantly occurs in the draining lymph nodes, the type
and number of innate immune cells that the vaccine initially interacts with at the administration site are
critical to the subsequent overall immune response.23,25. Vaccine administration causes tissue damage
and antigen depot formation at the injection site, which acts as a delivery system and leads to

controllable release of a number of endogenous molecules and antigens. Then, the damage/pathogen-
associated molecular pattern (DAMP/PAMP)–PRR pathways are activated.26,27. Immune and/or non-
immune cells at the injection site release inflammatory factors and recruit pioneer cells of the innate
immune system, the most important of which are macrophages and dendritic cells (DCs). The recruited
immune cells release cytokines and chemokines, which in turn recruit more immune cells, creating a
positive feedback loop.28.
2.2. The innate immune system
The innate immune cells capture and internalize antigens by PRRs; five types of PRR have been
identified: membrane-bound TLRs, C-type lectin receptors (CLRs), cytoplasmic NLRs, retinoic acid-
inducible gene-I-like receptors (RLRs), and several DNA sensors.29. The interaction between
DAMPs/PAMPs and PRRs leads to phagocytosis by the APCs. It has been reported that materials larger
than 500 nm are preferentially endocytosed by macrophages, while smaller materials are more often
phagocytosed by DCs.30. This process promotes the maturity and activation of APCs, which upregulate
the expression of the major histocompatibility complex (MHC) and co-stimulatory molecules, migrating
to the draining lymph nodes viavia afferent lymph vessels31 and initiating multiple downstream innate
immune pathways.32. Studies have shown that adjuvants in vaccines modulate the migration of immune
cells to the lymph nodes.25,33. Activated DCs serve as the link between innate and adaptive immunity,
triggering the adaptive immune response, presenting the antigen signal to T helper (Th) cells and
determining the type of subsequent immune response.34,35.
2.3. The adaptive immune system
Adaptive immunity can be categorized into two types: cell-mediated cytotoxic immunity and antibody-mediated
humoral immunity. The lymph node is a prime site for immune cell interactions.36. The differentiation and
maturation of T cells mainly occurs in the paracortex (T cell zone), where CD8 + T cells are primed in the central
paracortex and CD4+ T cells in the peripheral paracortex;37; the follicles (B cell zones) provide specific areas for
B cell maturation.38,39. Activated APCs present antigens to naïve T cells viavia MHC molecules. Naïve T cells
differentiate into different types of effector T cells, including Th1, Th2, Th17, T-regulatory cells (Treg) and
cytotoxic T lymphocyte (CTL).40,41. The cytokines secreted by mature APCs are essential for effective antigen-
specific T cell responses; these include Iinterleukin (IL)-12 for the activation of Th1 cells, IL-4 for Th2

activation, and IL-17 for Th17 activation. In turn, subsequent adaptive immunity is stimulated, including
triggering of antibody production by B cells, activation of cellular immunity and immune tolerance. At the same
time, memory T cells differentiate into the following three subtypes: central memory cells (TCM), effector
memory cells (TEM) and tissue-resident memory cells (TRM).42,43. The conditions under which memory T cells
differentiate are not only influenced by the host immune environment and the antigen (sustained low T cell
receptor signals, co-stimulation, and cytokines)43,44 The differentiation of memory B cells also results from many
factors, among which class-switch recombination (CSR) and somatic hypermutation (SHM) play a decisive role
in the differentiation bias towards plasma and memory B cells.45,46. The presence of memory B and T immune
cells is crucial for the permanent preventative or curative effects against a disease.47.
Induction of an efficacious specific immune response and maintenance of long-term immune memory with low
doses of antigen is area primary avenues of current vaccine research.
3. Composition of the vaccine delivery system
The effective introduction of delivery systems in novel vaccines plays a crucial role in enhancing and
optimizing vaccine efficacy. The relationship between the delivery system and adjuvant can be broadly
categorized into two groups: (1) the delivery systems with high immune stimulation can effectively
promote the host immune response of the loaded antigen. Although these delivery systems themselves
can act as adjuvants, further studies are required to evaluate their effectiveness, stability, repetitiveness,
and the long-term safety. (2) Vaccine delivery systems with low immune stimulation have excellent
ability to simultaneously deliver antigen and adjuvant into APCs in draining lymph nodes. These
delivery systems generally exhibit good biocompatibility with limited side effects. However, the
efficiencies of encapsulation and release of both antigens and adjuvants will impact the immune
system's vaccine response.
3.1. Delivery systems with high immune stimulation
Aluminum salts were first used as vaccine adjuvants since in 1926 48 and have been applicated in many licensed
human diseases vaccines for more than 90 years, such as diphtheria, tetanus, pertussis, hepatitis B, anthrax, and
influenza.16. The theory behind their use is that aluminum salts can provide a depot for antigen, resulting in
prolonging prolonged antigen release, promoting promoted the leakage of cytosolic “danger signals”, and
recruiting more inflammatory cells recruitment.49–51. Tremendous efforts were made to develop aluminum

adjuvants from micron-sized aggregates, predominantly inducing humoral immune response, to nanoparticles,
including γ-phase aluminum oxyhydroxide (γ-AlOOH),52, aluminum oxide (α-Al2O3),53, to a chemically
modified aluminum nanoparticles, such as by polyethyleneimine (PEI),54, increasing cross-presentation and
cellular immune response. Using aluminum salts in conjunction with TLR agonists is a current vaccine adjuvant
strategy, AS04 adjuvant, used in the human papillomavirus (HPV) vaccine Cervarix and the hepatitis B virus
(HBV) vaccine FENDrix, is a combination of aluminum salts and TLR4 agonists to rapidly activate local
immune responses and induce specific vaccine immunity.20,55. Similar inorganic materials, such as calcium
phosphate, mesoporous silica, and zinc oxide, have the potential for antigen delivery and adjuvant activity in
vaccines.56–60. Calcium phosphate has been used as an adjuvant in the diphtheria-–tetanus-–pertussis (DTP)–
polio vaccines.61. Other inorganic materials are still at the research stage and have not been used in a clinical
setting.
Emulsion systems have been developed as vaccine adjuvants and delivery systems since 1930, when
complete Freund’s adjuvant (CFA), a water-in-oil emulsion, was found to be able to carry an antigen
payload and enhance innate and adaptive immune responses.62. Incomplete Freund’s adjuvant (IFA),
which is formed by the removal of heat-killed mycobacteria from CFA, can also enhance immune
responses and reduce side effects.63. CFA and IFA are now rarely used in human vaccines with the
development of novel adjuvants and delivery systems. MF59, another oil-in-water formulation, is
widely used in influenza vaccines as an adjuvant and delivery system.17,18,64. In recent years, MF59
combined with a TLR agonist can not only enhance the immune response but also alter the type of
immune response elicited. For example, MF59 in formulation with TLR9 or TLR4 agonists, was found
to induce a Th1-biased immune response.65,66.
Virus-like particles (VLPs), including virosomes, are usually based on the highly organized and unified capsid
of a virus. The protein structures of VLPs have the same or highly related overall structures to the corresponding
native virus. The structure of viruses, presenting epitopes in dense repetitive arrays, make them suitable for the
delivery of various antigens.19,67. Viral capsids can also be modified by functional agents, which successfully
enhance target cell uptake and prolong the circulation time. Based on norovirus capsid shell (S) and protruding
(P) domains, two subviral nanoparticles (60-valent S60 and 24-valent P24) have been designed for use in vaccine
formulations, which have high immunogenicity.68. Tan and colleagues69 developed an icosahedral S60
nanoparticle coated with rotavirus protein 8* (VP8*), the major rotavirus-neutralizing antigen. The S60-–VP8*

nanoparticles significantly improved the humoral immune responses. This provided the theoretical basis for the
S60 nanoparticle as a functional platform for vaccine delivery loaded with other antigens. VLPs are highly
organized spheres, in which nucleic acid is packaged during their synthesis.70,71. VLP modifications can induce
immune responses at the cellular level. In an HPV-tumor mouse model, Hibma and colleagues72 discovered that
PADRE peptide-coated rabbit hemorrhagic disease virus (RHDV) VLPs could successfully enhance T cell
antitumor responses.
3.2. Delivery systems with low immune stimulation
Of these, the best known are liposomes, which have previously been approved for use as delivery systems for
the RTS,S/AS01 malaria vaccine.73. In the context of the COVID-19 pandemic, liposome-based mRNA vaccines
have also been developed at an unprecedented pace. Traditional vaccines using the inactivated SARS-CoV-2
causal agent of COVID-19 significantly improved the protection from infection; however, the mutated delta
variant, which became the predominant COVID-19 variant during the pandemic, was thought to be 55% to 90%
more transmissible than previous virus variants.74. mRNA vaccines have the potential to react to the mutated
virus, and antigen-independent methods render this approach faster and more efficacious than other
technologies. mRNA/lipid nanoparticle (LNP) technology is exemplified in this context by the Pfizer/BioNTech
BNT162b2 and Moderna mRNA-1273 vaccine candidates, both encoding the SARS-CoV-2 pre-fusion
stabilized S2P protein, which both showed very high efficacy in Phase 3 clinical trials.74–77. LNP has therefore
become a novel vaccine delivery platform, particularly as a carrier of DNA/mRNA. LNPs are approximately
100 nm in diameter, comprising four compounds: an ionizable lipid, cholesterol, PEGylated lipid, and a helper
lipid such as 1,2-distearoyl-sn-glyceroglycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glyceroglycero-3-
phosphoethanolamine (DOPE).78 The safety of LNP depends on the components of the biodegradable nontoxic
phospholipid bilayer shell, and the function can be adjusted by modifying the surface PEGylated lipid with
antibodies, small molecules, carbohydrates, peptides or protein; these modified LNPs are named ligand-targeted
liposomes. The PEGylated lipid component of LNPs consists of polyethylene glycol (PEG) conjugated to a
helper lipid, such as distearoyl phosphatidylethanolamine (DSPE). The molecular weight of the PEG can affect
the circulation time of DDSs, and PEGylation can also prevent the clearance of DDSs by the mononuclear
phagocytic system (MPS) or reticuloendothelial system (RES).79.
Polymeric materials are widely used as drug delivery systems owing to their biosafety, biodegradability, nearly
nontoxic nature and controllable release. Using various methods of polymeric particle injection, antigens can

successfully be presented to the APCs through prolonged release.80. Poly-D,L-lactate-co-glycolic acid (PLGA),
which hydrolyzes in vivo into biodegradable metabolite monomers, lactic acid, and glycolic acid, is a widely-
studied biodegradable delivery system that has been approved by the FDA, and acts as a copolymer in drug
delivery platforms. PLGA has also been studied as a vaccine delivery system for different pathogens, including
hepatitis B virus (HBV),81,82, Bacillus anthracis,83 and cancer.84.
Graphene oxide (GO) is another inorganic material used as a vaccine carrier owing to its biocompatibility and
outstanding capacity for delivery. Development in an alternative means of changing the surface character of GO
vaccines is required to overcome side effects caused by GO, including oxidative stress after vaccine injection.
Wang and colleagues85 devised a novel delivery system using lentinan-functionalized GO for antigen transport,
which improved the uptake of antigen, adaptive immunity, and long-term immune memory. Cui and
colleagues86 revealed that carnosine-modified GO could also mitigate the oxidative stress and inflammatory
responses induced by nanomaterials, and enhanced antigen-induced splenocyte reactivity in mice. As a vaccine
carrier, functionalized GO has significant adjuvant activity, activating cellular and humoral immunity. In the
future, it is expected to be used to improve the efficacy of vaccines.87.
4. Effects of physical properties of delivery systems on their function
For materials to be delivered accurately and efficiently, several obstacles need to be overcome; the barriers are
partly related to the means of administration, but there are some other common obstacles, including high
interstitial pressure impeding the entry of particles into tissues;88,89; elimination by the MPS or RES, in which
endocytosis of materials may be categorized into phagocytosis, macropinocytosis, clathrin-mediated
endocytosis, caveolae-dependent endocytosis, and independent endocytosis;90–92 lysosome degradation;93; and
the presence of nucleases in the case of DNA or RNA vaccines.
Vaccine delivery systems prolong the antigen circulation time;, improve the bioaccumulation in lymphoid
organs;, effectively target immune cells through the regulation of tunable physical properties;, enhance immune
effects, especially the specific immune responses;, shape the type of immune responses;, and significantly
reduce side-effects. Various physical properties of materials have important influence on these processes. The
effects of size, morphology, charge and elasticity on the action and mechanism of the delivery system are
discussed in the following sections and summarized in Table 2.

4.1. Size
Size is one of the most easily-controlled design parameters. Adjusting the size of materials can not only change
their pharmacokinetics and the circulation time of the vaccine payload but also have a significant impact on the
uptake of antigens by APCs, blood vessel transport, and the draining of lymph nodes and lymphoid organs, thus
changing the type of immune response elicited. Materials of different sizes are metabolized differently in vivo.
Smaller particles (<5 nm) can be excreted directly through the kidneys.94. Spherical particles 100–200 nm in
diameter have the potential to circulate for a long time in the blood.91. In addition, the route and retention time of
materials entering the draining lymph nodes also affect the efficacy of delivery systems. Optimizing the particle
size (<200 nm) can promote their entry into the draining lymph nodes.95. The interaction of Follicular dendritic
cells (FDC) with the materials determines their fate in the draining lymph node follicles to induce germinal
centers and long-term humoral immunity. Zhang96 previously indicated that small particles (5–15 nm) are
internalized and cleared by FDCs within 48 hours, and the fate of larger particles (50–100 nm) was quite
different, which aligned on the surface of FDC and retained for over 5 weeks (Figure Fig. 2A).
Different APCs have different selectivities for materials of different sizes. It is generally accepted that larger
particles (>500 nm) are preferentially taken up by macrophages, and smaller particles by DCs. The mode of
endocytosis also ultimately determines the fate of materials within cells,32, which is influenced by various
factors including cell type and particle characteristics.92. However, this section focuses only on the influence of
size on this process. Materials smaller than 30 nm generally cannot drive membrane wrapping and initiate
phagocytosis; caveolin-mediated endocytosis favors small materials less than 60 nm, and chathrinclathrin-
mediated endocytosis occurs with particles of approximately 120 nm. Macropinocytosis plays a major role in
endocytosis of materials larger than 1 μm.90,91,97. Jin and colleagues98 verified a deterministic kinetic model of
receptor-mediated endocytosis (RME) by using 130–660 nm DNA-wrapped single-walled carbon nanotubes
(DNA-SWNTs) and 14–100 nm Au nanoparticles (AuNPs), and found that both particle types had a maximum
endocytosis rate at a radius of approximately 25 nm when diffusion interactions were taken into account. They
also showed that exocytosis rate constants decreased with increasing nanoparticle or cluster size (Figure Fig.
2B).
Notably, micro-particles have some unique advantages in vaccine preparations. Biodegradable micro-particles
loaded with antigens can be used to create biomimetic artificial APCs, which have the advantage of flexibley
assembling assembly of the antigen, immune enhancers, molecular recognition elements and can inducing

induce potential specific immune responses.99. Liu and colleagues100 prepared poly(lactic acid) microparticles
(PLA-MPs) of uniform size, and verified their high antigen loading efficiency and ability to promote the
internalization of antigens by macrophages, which has have potential to improve vaccine delivery and adjuvants.
Hydrogels have hydrophilic and water-absorbing properties, and can thus expand in water and generate cross-
linking and mesh networks from hydrophilic polymers, forming micro- or nano-size polymer hydrogel
structures.101,102. Hydrogels have the advantage of high drug loading capacities and durable controlled release.103.
Meng and colleagues104 designed an ultrasound-responsive, self-healing hydrogel system, allowing the
controllable release of multiple nanovaccines in a single injection. Under ultrasonic treatment, the hydrogel and
solution state could be transformed. Accompanied with by the releasing of nanoparticles, DCs were stimulated
by antigens to initiate subsequent immune responses. In vivo, established tumors could be inhibited in
combination with an immune checkpoint blockade, with dramatically increasing in the percentages of tumor-
infiltrating CD8 + CTLs and NK1.1 cells. In contrast with to other synthetic materials, hydrogels have a three-
dimensional structure formed by porous and hydrated molecular networks, allowing them to provide space for a
variety of immune cells, a property that is similar to the natural tissue microenvironment. Mooney and
colleagues105 used alginate hydrogels to provide an immune niche for immune cell infiltration, which controlled
the release of granulocyte-macrophage colony stimulating factor (GM-CSF). The polymer allowed the
spontaneous and continuous release of GM-SCF, and subsequently induced DC recruitment but did not induce
DC maturation and activation. Yang and colleagues106 developed a vaccine nodule, comprising anti-programmed
cell death protein 1 (anti-PD1) in a self-assembled peptide-based nanofibrous hydrogel (RADA16), resulting in
enhanced T cell immunity, thus holding promise for immunotherapy against cancer and infection.
4.2. Morphology
Morphology is another important controllable parameter of materials. Understanding the relationship between
materials morphology and cellular responses (in both immune and non-immune cells) is essential to developing
effective immune adjuvants and vaccine delivery systems.24. The morphology of materials modulates not only
the efficiency but also the means of uptake by immune and non-immune cells. Under the same diameter, the
efficiency of endocytosis for cylindrical morphologies is higher than spherical.107. Some studies have shown that
long-rod materials are more rapidly internalized and have a stronger activation effect on mouse bone marrow-
derived dendritic cell (BMDC) maturation (CD86/CD80 expression) and the production of cytokines (IL-1β and
IL-6) than short-rod and spherical morphologies (Figure Fig. 3A, and B).108,109. However, other studies have

shown that spherical materials are more readily ingested than rods by non-phagocyte cells.110,111. It has been
indicated that the membrane encapsulation efficiency is related to the contact area between materials and cells,
and the competition between the diffusion kinetics of the receptor and the thermodynamic driving force.112. The
receptor-mediated membrane wrapping process of elastic NPs with different sizes and shapes has been
systematically investigated using a coarse-grained molecular dynamic (CGMD) simulation. The results showed
that under the same bending constant, the wrapping efficiency of oblate NPs was better than spherical and
prolate NPs (Figure Fig. 3C and, D). In terms of the means of uptake by cells, clathrin-mediated endocytosis is
the most common form of nonspecific uptake;92; rods activate caveolae-dependent endocytosis,113, and triangle
morphologies may enter cells viavia macropinocytosis and phagocytosis.114. Different cells have different
responses to the material morphology; for example, epithelial cells show different interaction efficiencies to
gold rods with different aspect ratios, while the uptake efficiency of macrophages is not affected by this
parameter.115.
The way in which material morphology influences the type of immune response remains controversial. Rashad
and colleagues116 suggested that nanofibers enhanced uptake into DCs and the Th1 immune response,
accompanied by the high production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and antigen-specific
immunoglobulin G (IgG), while nanospheres tend to induce Th2 immune responses. However, Sunny and
colleagues117 came to the opposite conclusion. In vivo small spherical carboxylated polystyrene materials (193
nm) generated Th1 and Th2 immune responses, whereas rod-shaped ones (1530 nm) produced stronger Th2-
biased immunity. Therefore, the morphology does not act alone to determine the efficiency and the means of
uptake, but is complementary to other physical properties, cell types in contact with the materials, and biological
factors such as the surrounding environment, which jointly determine the properties and functions of vaccine
delivery systems.
4.3. Surface charge
The surface charge of materials affects their internalization by immune and non-immune cells, and their
biodistribution. In terms of surface charge alone, cationic materials are thought to easily endocytose in
comparison to anionic and neutral particles.90,118–121. For vaccines, the tendency to be internalized is conducive to
uptake by APCs and thus immune function; however, this is also a primary reason for shorter circulatory
timeframes. There are many factors affecting the surface charge of materials, including the biomolecules being

carried and the biological environment. When particles aggregate in vivo, especially in blood circulation, a
‘protein corona’ forms on the surface. Nanoparticles are rapidly covered by selected biomolecules, such as
coagulation, immunoglobulin, apolipoprotein, and complement.122,123. The composition of ‘protein corona’ is not
only affected by the material, size, and surface modification of nanoparticles, but also by the complex biological
environment.124. The surface charge usually decides the inner protein corona also called hard protein corona,
which changing the surface properties and cellular uptake of the material in vivo.125–127 Changes in the biological
microenvironment also alter the properties of materials affected by surface charge, such as the presence of a
dense extracellular matrix and abnormal lymphatic vessels in the tumor microenvironment, leading to reduced
permeability of the material, especially for cationic particles.128. Sun and colleagues129 constructed poly(ethylene
glycol)-block-poly(D,L-lactide) (PEG-b-PLA) nanoparticles coated with different lipids to yield materials with
positive, neutral or negative charges; the cationic nanoparticles had an inferior blood circulation time compared
with the anionic and neutral particles, but outperformed both in terms of tumor penetration and tumor growth
suppression (Figure Fig. 4A–C). To develop targeted and effective vaccines, the design of materials with
conditional responsive charge conversion may overcome the obstacles presented in this section. This kind of
delivery system is required to ‘switch off’ internalized antigens until the target organ is reached, also known as
controlled release.130. Using charge properties, a mannosylated zwitterionic-based cationic liposome system
(man-ZCL) was constructed by Qian and colleagues131 based on the endosomal/lysosomal escape ability of
zwitterionic lipid distearoyl phosphoethanol-amine-polycarboxybetaine (DSPE-PCB). This system was able to
compound DNA antigens, forming a tight structure to protect it from ribozyme degradation in vivo, which
significantly enhanced anti-human immunodeficiency virus (HIV) immune responses with low toxicity, and
induced Th1/Th2 mixed immunity (Figure Fig. 4D, and E).
4.4. Elasticity
Apart from the size, morphology and surface charge of materials, elasticity has recently become a major focus
as a parameter affecting internalization in APCs, especially macrophages and DCs. Elasticity can be also
explained as softness, which is mainly calculated by a using Young’s modulus. Soft particles (100 kPa) have
been found to interact with macrophages only briefly, and rigid materials (10 MPa) yield up to five times more
efficient cellular uptake by bone marrow derived monocytes (BMDMs) compared with soft nanomaterials.
Softness acts in conjunction with morphologies and size to determine the internalization of particles.132.
Elasticity not only affects the interaction of particles with phagocytes such as macrophages but also has an effect

on uptake by cancer cells. Hui133 showed that the elasticity of particles affects the type of interactions between
particles and cells, in turn affecting the efficiency of particle internalization by the cells. The receptor-–ligand
reaction and membrane encapsulation that occur during particle uptake can also deform softer particles,
affecting the endocytosis of softer particles (Figure Fig. 5A–C). However, this does not necessarily mean that
rigid nanomaterials have a superior uptake efficiency. In another study, Xia and colleagues134 created a PLGA
nanoparticle-stabilized Pickering emulsion adjuvant system (PPAS), indicating a high antigen-loading capacity
and an amplified mechanosensory ability. The pliability and lateral mobility of this antigen-loaded Pickering
emulsion were important factors in enhancing the efficacy of vaccination especially in protection against B16-
mucin 1 peptide (B16-MUC1) and influenza virus compared with aluminum salts (Figure Fig. 5D and, E).
5. Perspectives
The development of vaccinations has occurred over a long time, during which different components of vaccines,
including antigens, adjuvants and delivery systems, have been developed. The introduction of novel delivery
systems has further optimized vaccine development. At present, research into vaccine delivery systems can be
usefully divided into two parts. First, the mechanism of action of the existing delivery system of the vaccine
should be further clarified and optimized. An in-depth understanding of the mechanism, optimizing the means of
administration, and reducing toxic and side effects are important for the application of the vaccines in the
general population. Second, the development of novel vaccine delivery technologies is based on a full
understanding and analysis of current unmet needs, and the impact of the physical properties of materials on
their mechanism, the interactions between materials and the host immune system. When we pursue the
efficiency and specificity, adequate safety is still a primary requirement of human vaccines. For example, the a
high oil content does not meet the safety or quality standards for human vaccine. The ideal vaccines should be
simple in composition, provide adequate immune protection and long-term efficacy against the diseases they
aim to protect against, provide protection against mutant variants of the causal agent, and be administered in a
non-invasive way. To achieve this goal, concerted efforts and cooperation among various industries are still
needed.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements

This work was supported by grants from the National Key Research and Development Program of
China (2017YFA0208100), NSFC (32171379, 91642208, 81871478, and 81422026), Jilin Scientific
and Technological Development Program (20190201094JC, and 20200301007RQ), Interdisciplinary
Innovation Project of the First Hospital of Jilin University (JDYYJCHX001), and the Fundamental
Research Funds for the Central Universities, JLU.
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Fig.Figure 1 Mechanism of vaccines. Tissue damage and depot formation occurs at the injection site after
vaccine administration viavia different routes, resulting in the recruitment of cells of the innate immune system.
In draining lymph nodes (dLNs), the activated antigen-presenting cells (APCs) present antigens to naïve T and
B cells, initiating adaptive immunity and leading to the generation of mature and memory immune cells with
various executive functions. (IFN: interferon, TNF: tumor necrosis factor, IL: interleukin).
Fig.Figure 2 The interaction of antigen presenting cells (APCs) with nanoparticles (NPs) of different sizes. (A)
Small particles (5–15 nm in size) are internalized and cleared by follicular dendritic cells (FDCs) within 48
hours; the fate of larger particles (50–100 nm) is quite different, with retention for over 5 weeks (aligned on the
surface of FDCs.96. (B) Single-walled carbon nanotubes (SWNTs) and Au nanoparticles (AuNPs) have a
maximum endocytotic rate at a radius of approximately 25 nm, verified by the deterministic kinetic model of
receptor-mediated endocytosis (RME).98. Reprinted from ref. 96 with the permission from American Chemical
Society, copyright 2019; Reprinted from ref. 98 with the permission from American Chemical Society,
copyright 2009.
Fig.Figure 3 The effect of nanoparticle (NP) morphology on NP–cell interaction. (A) Representative TEM
images of AlOOH nanorods obtained after a synthesis period of 2 h (Rod 1), 3 h (Rod 2) and 24 h (Rod 5). (B)
Long rod particles had a stronger activating effect on bone marrow derived cell (BMDC) maturation and
production of cytokines than short rod and spherical particles.109. (C, and D) Under the same bending constant,
the NP morphology plays a significant role. Membrane wrapping efficiency for the different morphologies of
NP is ranked as follows: oblate > spherical > prolate.112. Reprinted from ref. 109 with the permission from

American Chemical Society, copyright 2013; reprinted from ref. 112 with the permission from American
Chemical Society, copyright 2019.
Fig.Figure 4 Application of cationic nanoparticle (NP) systems in vaccines. (A) Poly(ethylene glycol)-block-
poly(D,L-lactide) (PEG-b-PLA) can be tuned by various lipids to yield three groups of nanoparticles with
positive, neutral or negative charges. Cationic PEGylated nanoparticles had better tumor penetration (B) and
suppressed tumor growth (C).129. (D) Using the particle charge properties, a mannosylated zwitterionic-based
cationic liposome system (man-ZCL) was constructed. (E) Man-ZCL0.4/5 enhanced human immunodeficiency
virus (HIV) DNA Env-specific T cell proportion and all groups treated with man-ZCL lipoplexes exhibited a T
helper type (Th) 1/Th2 mixed immune responses (1 < Th1/Th2 < 2).131. Reprinted from ref. 129 with the
permission from Elsevier, copyright 2016; reprinted from ref. 131 with the permission from Elsevier, copyright
2016.
Fig.Figure 5 Effects of nanoparticle (NP) elasticity on cellular interactions. (A) Cellular uptake and binding of
silica nanocapsules (SNCs) were measured at 4 °C℃ and 37 °C℃. Both the uptake and binding of SNCs by
macrophages and SKOV3 (human ovarian adenocarcinoma cells) increased with SNC elasticity. During uptake,
stiff SNCs remained spherical (B, top) and soft SNCs were deformed (B, bottom). SNC morphological changes
played an important role in modulating cellular uptake (C).133. A poly-(lactic-co-glycolic acid) (PLGA)
nanoparticle-stabilized Pickering emulsion adjuvant system (PPAS) with an amplified mechanosensory ability
enhanced antigen uptake (D) and functioned as a potent adjuvant for an intranasal lethal dose of influenza
(A/FM/1/47, H1N1) and mucin 1 peptide (MUC1) anti-tumor vaccines (E).134. Reprinted from ref. 133 with the
permission from American Association for the Advancement of Science, copyright 2020; reprinted from ref.
134 with the permission from Macmillan Publishers Limited, part of Springer Nature, copyright 2018.
Table 1 Summary of common types of vaccines
Vaccine type Description Strength of Limitations Example
immune response vaccinations
Inactivated Inactivated Inactivated People may need Hepatitis A, Fflu,
vaccines use a vaccines do not several doses over Ppolio, Rrabies
killed version of usually provide time (booster

the causal immune vaccines) in order to
pathogen as an protection that is obtain ongoing
antigen as strong as live immunity against the
vaccines disease
Live-attenuated Live vaccines This kind of Not suitable for Measles,
use a weakened vaccine creates a immunocompromised Mmumps,
(or attenuated) strong and long- patients Rrubella,
form of the lasting immune Rrotavirus,

This vaccine must be
causal pathogen response Ssmallpox,
stored at low
as an antigen Cchickenpox,
temperatures
Yyellow fever
mRNA mRNA vaccines mRNA itself has This vaccine must be COVID-19
stimulate the limited stored at low
production of immunogenicity; temperatures
proteins by the the protein
recipient in translated by the
order to trigger recipient may
an immune elicit a very
response strong immune
response
Subunit, Subunit, These vaccines People may need Hib (Hhemophilus
recombinant, recombinant, generally several doses over influenzae type B)
polysaccharide, polysaccharide, produce a very time (booster disease, Hhepatitis

and conjugate and conjugate strong immune vaccines) in order to B, Hhuman
vaccines use response that is obtain ongoing papilloma virus
specific pieces targeted to key immunity against the (HPV),
of the pathogen, parts of the disease Ppneumococcal
such as protein, pathogen disease,
sugar, or capsid Mmeningitis,
(casing) Sshingles
Toxoid Toxoid vaccines Toxoid vaccines People may need Diphtheria;
use a toxin do not usually several doses over Ttetanus
(harmful provide time (booster
product) made immunity vaccines) in order to
by the pathogen (protection) that obtain ongoing
that causes a is as strong as immunity against the
disease live vaccines disease
Viral vector Viral vector Viral vaccines This type of vaccine COVID-19, Zzika,
vaccines use a are easily raises concerns about Fflu, Hhuman
modified recognized by the virus integration with immunodeficiency
version of a host immune the genome of the virus (HIV)
different virus system and elicit recipient
as a vector to a strong immune
deliver response
protection
Table 2 Summary of the effects of materials physical properties on their functions

Materials Model Immunological effects
Effects of size
CdSe/ZnS Renal filtration threshold Hydrodynamic diameter smaller
than 5.5 nm results in rapid and
efficient urinary excretion94
AuNPs Lymph node follicles Follicular dendritic cells clear
small particles (5–15 nm) within
48 h, and retain larger ones (50–
100 nm) for over five weeks96
DNA-SWNTs, AuNPs, PLGAs Deterministic kinetic model Both SWNT and AuNP have a
maximum endocytotic rate at a
radius of approximately 25 nm
radius98
PLGAs, PVAs Melanoma Biodegradable microparticles
loaded with antigens can be
used to make biomimetic
artificial antigen-presenting
cells99
PLA-MPs RAW264.7 PLA-MPs have a high antigen
loading efficiency and an ability
to promote the internalization of
antigens by macrophages100
Nanocomposites Melanoma Ultrasound-mediated gels
provide controllable and durable

vaccine delivery104
Alginate gels — Alginate gels provide a
spontaneous and continuous
release of GM-SCF and induce
dendritic cell recruitment105
Nanofibrous gels Lymphoma Self-assembled nanofibrous gels
enhance T cell immunity106
Morphology
Mesoporous silica nanoparticles A375 cell MSNs with larger aspect ratios
(MSNs) were taken up in larger amounts
and had faster internalization
rates108
AlOOH nanorods THP-1 cell Long rods stimulate greater
NLRP3 inflammasome
BMDCs
activation and IL-1β
production109
Polystyrene (PS) NPs HelaHeLa cell The cellular uptake of spherical
PS NPs is greater than rod-
shaped particles110
AuNPs HelaHeLa cell Spheres have a higher
probability of entering the cell
compared with rods111

MSNs Caco-2 cell More efficient cellular uptake is
observed with rods than
spheres113
AuNPs RAW264.7 A higher cellular uptake is
observed with triangles than
rods or stars114
Gold nanorods (GNRs) A549 cell Epithelial cells have different
interaction efficiencies with

HelaHeLa cell
gold rods with different aspect
J774A.1 cell ratios, while the uptake
efficiency of macrophages is not
affected by the aspect ratio115
Peptide amphiphile (PA)/ODNs Balb/c mice Nanofibrous morphologies
enhance Th1 immune responses,
while nanospheres tend to
induce Th2 immune responses116
Carboxylated polystyrene Balb/c mice Spherical particles (193 nm)
particles generate Th1 and Th2 immune
responses, while rods (1530 nm)
stimulate stronger Th2-biased
immunity117
Charge
Hydroxyapatite (HAP) NPs MC3T3-E1 cell HAP NPs with positive surface

charges have superior
biocompatibility and higher
cellular uptake in osteoblast cell
line compared with those with
negative charges118
Amine-containing hydrogels Alveolar macrophages (AM) Cationic particles increase AM
association and promote lung
dendritic cell (DC) maturation119
AuNPs HelaHeLa cell When particles aggregate in
vivo, a ‘protein corona’ changes

RAW264.7
the surface properties and
bEnd.3 cell cellular uptake of the
materials126
Silica and polystyrene NPs Quantitative proteomic data When NPs enter the
physiological environment,
protein coronas rapidly establish
and modulate the
pathobiological effects127
PEG-b-PLA NPs Tumor model Cationic PEGylated
nanoparticles revealed better
tumor penetration and
suppressed tumor growth
compared with others129

Elasticity
Discoidal polymeric RAW264.7 Rigid materials promote more
nanoconstructs (DPNs) efficient cellular uptake by bone

BMDMs
marrow derived macrophages
compared with soft materials132
SNCs RAW264.7 The elasticity of particles affects
the types of interactions

SKOV3 cell
between particles and cells, and
thus the efficiency of their
internalization by those cells133
Pickering emulsion Lymphoma Pliability and lateral mobility of
antigen-loaded Pickering
Melanoma
emulsion are important factors
Influenza in enhancing vaccination134

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<<Please check the yellow highlights>>BM|d2bm00011c|10.1039/d2bm00011c|REV

Engineering optimal vaccination strategies: effects of physical properties of the delivery

Ge Zhu,a,b Yong-Guang Yanga,b,c and Tianmeng Sun*a,b,c

Received 5th January 2022, Accepted 23rd January 2022

DDSs on the success of vaccines.

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optimized and suitable vaccine delivery systems in the future.

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primary goals of research on contemporary vaccines.

and have satisfactory efficacy.20,21.

2.1. Injection site reactions

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positive feedback loop.28.

2.2. The innate immune system

determining the type of subsequent immune response.34,35.

2.3. The adaptive immune system

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doses of antigen is area primary avenues of current vaccine research.

3. Composition of the vaccine delivery system

system's vaccine response.

3.1. Delivery systems with high immune stimulation

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including γ-phase aluminum oxyhydroxide (γ-AlOOH),52, aluminum oxide (α-Al2O3),53, to a chemically

modified aluminum nanoparticles, such as by polyethyleneimine (PEI),54, increasing cross-presentation and

to induce a Th1-biased immune response.65,66.

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3.2. Delivery systems with low immune stimulation

lipid such as 1,2-distearoyl-sn-glyceroglycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glyceroglycero-3-

phagocytic system (MPS) or reticuloendothelial system (RES).79.

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hepatitis B virus (HBV),81,82, Bacillus anthracis,83 and cancer.84.

future, it is expected to be used to improve the efficacy of vaccines.87.

4. Effects of physical properties of delivery systems on their function

endocytosis of materials may be categorized into phagocytosis, macropinocytosis, clathrin-mediated

endocytosis, caveolae-dependent endocytosis, and independent endocytosis;90–92 lysosome degradation;93; and

the presence of nucleases in the case of DNA or RNA vaccines.

discussed in the following sections and summarized in Table 2.

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receptor-mediated endocytosis (RME) by using 130–660 nm DNA-wrapped single-walled carbon nanotubes

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4.3. Surface charge

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