X. He et al.
Journal of Controlled Release 337 (2021) 306–316
is based on supramolecular assembly of sequence specific polymers,
such as nucleic acids, proteins and peptides. Among them, peptides have
attracted much attention due to their biocompatibility and chemical
versatility. [37] They have been employed as building blocks to
construct different supramolecular nanomaterials for a wide range of
potential applications. Hydrogels formed by peptide self-assembly could
apply for immunomodulation, inducing antigen specific humoral im-
munity and cellular immune response. [42] However, the requirement
of covalent conjugation of antigen or the introduction of additional
heating-cooling process hinders the further development of peptide
derivatives for immunotherapy. In addition, it has been shown that the
short peptide composed of two or three amino acids can be used as a
powerful self-assembly motif. [43] For example, the well-known
diphenylalanine peptide has been widely used to prepare various su-
pramolecular self-assembled nanomaterials. [44,45] Nevertheless, non-
aqueous solvents are not only an essential condition for the self-
assembly of short peptides, but also exert a significant impact on the
nanoscale morphology of short peptide self-assemblies, which seriously
hinders their applications in drug delivery. At the same time, there are
few reports on the supramolecular self-assembled functional nano-
materials based on single amino acid. Compared with peptides, amino
acids have simpler structure, better biocompatibility, repeatability and
operability, therefore, they have greater attraction and potential in the
self-assembly of functional supramolecular nanocarriers for drug de-
livery. [46,47]. Therefore, in this study, we designed and screened a
series of clickable amino acid derivatives as building blocks of functional
supramolecular self-assembly nanoparticles to realize the intracellular
delivery of protein antigen and oligonucleotide adjuvant into the same
DCs to enhance the immune response. The traditional amino acid pro-
tecting group Fmoc was changed to DIBO functional group for copper
free click chemistry, which can efficiently expand the types of self-
assembled materials under mild conditions, strengthen the self-
assembly behavior and avoid any non-aqueous solutions that might
inactivate protein and nucleic acid. TEM and DLS experiments revealed
that self-assembled nanoparticles can be easily formed with size of about
the DC maturation and antigen cross-presentation in mice by increasing
+
+
+
+
the percentages of CD11c SIINFEKL DCs and CD86 CD80 matured
DCs. In addition, high secretion of immunostimulatory cytokines
including TNF-
α, IFN-γ and IL-6 were also found in the serum of mice
treated with DA6C1/OVA/CpG nanovaccine. Moreover, this nano-
vaccine could also increase the production of anti-OVA IgG titres in the
+
serum of mice, enhance the activation of antigen-specific CD8 and
+
CD4 T cells, and promote the prolifieration of splenocytes. Therefore,
the strong in vivo immune responses induced by DA6C1/OVA/CpG
nanovaccine demenstrated its promising role in prevention and treat-
ment of cancer.
After vaccination by using an E.G7-OVA tumor model, DA6C1/OVA/
CpG nanovaccine exhibited superior to other formulations in delaying
tumor development and improving survival rate. The treatment of
established tumors showed that DA6C1/OVA/CpG nanovaccine signif-
icantly inhibited tumor progression and induced effective CTL infiltra-
tion. More importantly, DA6C1/OVA/CpG nanovaccine combined with
anti-PD1 therapy was more effective in alleviating tumor immunosup-
pression, increasing the proliferation and infiltration of CTLs, thus
leading to better anti-tumor treatment effect. Taken together, these re-
sults demonstrated the great potential of clickable amphiphilic amino
acid derivatives tuned self-assembly of antigen and adjuvant as a
convenient and powerful strategy for the development of nanovaccine to
improve cancer immunotherapy.
4. Materials and methods
4.1. Materials
6-aminohexan-ol and 10-aminodecan-1-ol were purchased from
9dingchemistry (Shanghai, China). L-arginine and D-arginine was pur-
chased from Alfa Aesar (Shanghai, China). Other chemical reagents
were bought from energy chemical (Beijing, China). Ovalbumin (OVA)
′
was obtained from Sangon Technology (Shanghai, China). CpG (5 -TCC
′
′
ATG ACG TTC CTG ATG C-3 ) and Rhodamine-labeled CpG-ROD (5 -
′
1
00 nm after dissolving these amphiphilic arginine derivatives into
Rhodamine-TCC ATG ACG TTC CTG ATG-3 ) were synthesized by San-
aqueous solution. Agarose gel retention assay indicated that OVA can be
incorporated into the structure of self-assembled nanoparticles, and the
introduction of cross-linking agents can further enhance the loading
capability of these nanoparticles. Through in vitro combinatorial
screening, we found that self-assembled nanoparticles based on DA6C1
had high antigen and adjuvant loading capacity and cellular endocytosis
efficiency, indicating that nanovaccines based on DA6C1 tuned self-
assembly has great potential in immunotherapy.
gon Technology (Shanghai, China). DAPI was purchased from solarbio
(Beijing, China). And, TNF-
α, IL-6, IFN-γ ELISA Kit was also obtained
from solarbio (Beijing, China). Fluorescence labeled CD11c, CD80,
CD86, SIINFEKL/H-2 kb, CD3, CD4, CD8, Foxp3 antibody, FITC-
conjugated antimouse IFN-γ and HRP-conjugated antimouse IgG were
obtained from Biolegend (San Diego, USA). Recombinant mouse GM-
CSF and IL-4 were purchased from PeproTech (Rocky Hill, USA). RAW
264.7 cell line was bought from National Infrastructure of Cell Line
Resource (Beijing, China). E.G7-OVA cell line was purchased from Bio-
leaf (Shanghai, China).
As an important type of APCs, DCs play a crucial role in inducing
adaptive immunity by activating T cells. Nanovaccines can greatly
determine the efficiency of antigen uptake and intracellular localization,
thus affecting the specific immune response and protective effect. The
confocal fluorescence images of BMDCs incubated with OVA and CpG
co-loaded DA6C1 nanovaccines demonstrated that DA6C1 could
significantly enhance the cellular uptake of OVA and CpG, and inter-
nalize them into the same DCs, which would be beneficial to induce
stronger immune response and more effective immune activity. The
results showed that DA6C1/OVA/CpG nanovaccine could significantly
improve the efficiency of antigen cross presentation and DC activation
4.2. Agarose gel retention assay
OVA (8 g) were respectively mixed with DA1, DA2, DA3, DA4, DA5
μ
and DA6 at a certain mass ratio of 1:4. Then crosslinking agents C1-C3
were respectively added to the mixture at a certain molar ratios. For
C1 and C2, the molar ratio of C1 or C2 to DA1–6 was 1:3, for C3, the
molar ratio of C3 to DA1–6 was 1:4. All samples were subjected to 1 h
electrophoresis on 1% agarose gel and then the images were collected by
gel imaging instrument (Gel Doc Bio-Rad).
(
Fig. 4C-F), while free OVA+CpG had little effect on the stimulation of
DC maturation, which was consistent with the fact that naked negative
charged antigen and nucleic acid can hardly enter the cytoplasm and
were also easy to be degraded by enzymes. Moreover, this nanovaccine
4.3. Uptake of OVA-FITC by RAW264.7 cells
could also produce high levels of TNF-
α
and IL-6 (Fig. 4G and H), which
Cellular uptake studies were performed in RAW264.7 cells at an
4
are cytokines highly related to antitumor immunity. Overall, these re-
sults suggested that the nanovaccine based on DA6C1 tuned self-
assembly of OVA and CpG can greatly enhance the immune response,
thus becoming an effective method for DC activation. In addition, we
evaluated the abilities of these vaccines to improve DC maturation in
vivo. DA6C1 based various nanovaccines could significantly improve
initial cell density of 1 × 10 in 96-well plate. After overnight incuba-
tion, cells were treated with various OVA-FITC nanoformulations and
incubated for another 8 h. The cells were then slowly washed with PBS
and stained with DAPI after fixing with paraformaldehyde. Cell images
were acquired by fluorescence microscopy (Olympus IX 51). After that,
the cells were trypsinized, centrifuged and resuspended in PBS for flow
3
13