Volume 10
Number 14
14 April 2022
Pages 2263–2752

Journal of

Materials Chemistry B
Materials for biology and medicine
rsc.li/materials-b

Themed issue: Bioinspired Surfaces Engineering of Biomaterials

Chie Kojima et al.
Carboxy-terminal dendrimers with phenylalanine
for a pH-sensitive delivery system into immune cells
including T cells

I

PAPER

M nde
ed xe
lin d ine!

ISSN 2050-750X

Journal of

Materials Chemistry B
View Article Online

PAPER

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Cite this: J. Mater. Chem. B, 2022,
10, 2463

View Journal | View Issue

Carboxy-terminal dendrimers with phenylalanine
for a pH-sensitive delivery system into immune
cells including T cells†
Hiroya Shiba,a Misaki Nishio,a Mei Sawada,a Mamiko Tamaki,a Masataka Michigami,bShinya Nakai,b Ikuhiko Nakase, b Ikuo Fujii,b Akikazu Matsumotoa andChie Kojima *a
Although T cells play important roles in various immune reactions, there are only a few reports ondelivery systems into T cells. Our previous study showed that carboxy-terminal phenylalanine (Phe)modified polyamidoamine (PAMAM) dendrimers have both temperature- and pH-sensitive properties,
which are aﬀected by the chemical structure. The self-assembled structures of Phe, observed inphenylketonuria, enhance the protein aggregation, the association with the cell membrane and themembrane permeability. In this study, we applied the Phe-modified dendrimers to a pH-sensitive drugdelivery system into T cells. Dendrimers with diﬀerent amino acids and acid anhydrides weresynthesized, and their pH-responsive association with T cells and their subsets was investigated. The
dendrimers modified with Phe and cyclohexanedicarboxylic acid (CHex) showed higher uptake into

Received 9th September 2021,
Accepted 4th December 2021

various cells, including Jurkat cells, CD3+ T cells, CD3 + CD4+ helper T cells and CD3 + CD8+ killer
T cells. These dendrimers were internalized into T cells via endocytosis, and their cellular uptake was

DOI: 10.1039/d1tb01980eenhanced under weak acidic conditions (pH 6.5). Our results showed that Phe- and CHex-modified
dendrimers have a delivery potential to T cells and their subsets, which may be useful for cancer

rsc.li/materials-b

immunotherapy.

1. Introduction
The application of stimuli-responsive materials to drug delivery
systems (DDS) is of great importance, since these materials
change their properties when they are exposed to external
stimuli, such as temperature, ultrasonication, and magnetic
fields, and internal stimuli, such as pH, reduction environments, and environment-specific enzymes.1 pH-responsive DDS
have been studied by many researchers because anaerobic
glycolysis produces lactic acid in tumor tissues, which creates
acidic tumor microenvironments.2 In addition, cells endocytose extracellular substances, whose late endosomes and lysosomes become acidic.2 Therefore, pH-sensitive DDS is usefulfor tumor-targeted drug delivery as well as for endosomal
escape.2 Dendrimers are highly branched macromolecules withwell-defined structures, and their unique properties diﬀer froma

Department of Applied Chemistry, Graduate School of Engineering, Osaka
Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
E-mail: kojima@chem.osakafu-u.ac.jp; Fax: +81 72 254 8190;
Tel: +81 72 254 8190b
Department of Biological Science, Graduate School of Science, Osaka Prefecture
University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1tb01980e

This journal is © The Royal Society of Chemistry 2022

those of linear polymers. Since dendrimers can encapsulate
and/or modify various bioactive compounds, such as drug
molecules, they are potent nanoplatforms for DDS. Polyamidoamine (PAMAM) dendrimers, which are commercially available, have a large number of inner tertiary amino groups, which
can work as pH-sensitive units. Acidic compounds can easily beencapsulated due to their electrostatic interactions with tertiaryamino groups, and the binding aﬃnity increases with thedecrease in pH.3
Our previous report indicates that the amino-terminal
PAMAM dendrimers with phenylalanine (Phe) have temperature- and pH-responsive properties in the ranges larger thanpH 7.4 Also, carboxy-terminal Phe-bearing PAMAM dendrimers,
which were synthesized by reacting with Phe and succinyl
anhydride (Suc), showed temperature- and pH-responsive properties in a diﬀerent manner.5,6 A PAMAM dendrimer modified
with Suc and Phe in this order (PAMAM-Suc-Phe) showed UCSTtype thermosensitivity at pH 6,5 but the PAMAM dendrimer
modified with Phe and Suc in this order (PAMAM-Phe-Suc)
showed both LCST- and UCST-type thermosensitivity at pH 4
and 6, respectively.6 Phenylketonuria (PKU) is a metabolic
disorder disease which induces an elevated concentration of
Phe.7 Fibril structures are formed in a high concentration of

J. Mater. Chem. B, 2022, 10, 2463–2470 | 2463

View Article Online

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Paper
Phe because of the self-assembly. The self-assembled structuresof Phe enhance the protein aggregation, the association withthe cell membrane and the membrane permeability, which are
known to be the cause of PKU.7 These inspired us to use Phemodified dendrimers as a potent drug carrier with pH-sensitive
properties.
Cancer immunotherapy is a cancer treatment method that
attacks tumor cells by activating the immune systems ofpatients.8 Immune surveillance against cancer comprises various kinds of immune cells, in which T cells play importantroles. T cells are classified into various kinds of subsets. CD4and CD8-positive T cells are known as helper T cells andcytotoxic T cells, respectively. Helper and cytotoxic T cells
control immune reactions and induce cell death, respectively.
These subsets are a possible target in cancer immunotherapy. It
has been reported that lactate produced in tumor tissues
impairs the migration of T cells by reducing the expression ofchemokine receptors. Also, excess lactate regulates macrophage
polarization and suppresses the functions of killer cells.9 Thus,
the upregulation of T cells in acidic tumor microenvironments
is important for eﬃcient cancer immunotherapy. T cells areactivated by mediating with phagocytic immune cells, such as
macrophages and dendritic cells,10 because direct delivery into
T cells is diﬃcult. There are many reports on pH-sensitive DDS
into antigen-presenting cells, in which endosomal escape canbe induced.10 However, there are only a few reports of delivery
systems to T cells.11,12 Schmid et al. and Cao et al. developed
CD8- and CD4-conjugated poly(lactic-co-glycolic acid) (PLGA)
nanoparticles for T cell-targeted delivery, respectively.11 They
were used for the antibodies that can be targeted at the cellsurface of T cells. Because eﬃcient delivery into T cells using
antibodies is diﬃcult, the delivery systems into T cells still need
to be developed.
We previously reported that the anionic terminal PAMAM
dendrimers whose generation is greater than generation-4 (G4) wereeﬃciently accumulated in lymph nodes via intradermal injection.13
The sentinel lymph node is the first organ to drain malignant tumor
cells. Lymph node-targeted delivery, especially delivery into lymph
node-resident T cells, is necessary for cancer immunotherapy.
Previously, 2-carboxy-cyclohexanolated (CHex) PAMAM dendrimer
with Phe, PAMAM-CHex-Phe, was well associated with lymph noderesident lymphocytes, including T cells.14 In this study, we
attempted to construct a pH-sensitive delivery system into T cells
by using carboxy-terminal Phe- and CHex-modified dendrimers with
diﬀerent structures, PAMAM-CHex-Phe and PAMAM-Phe-CHex.
Other types of hydrophobic carboxy-terminal dendrimers were alsosynthesized by changing the amino acid (leucine (Leu)), acidanhydrides, and reaction order (Fig. 1), to investigate the relationship between the dendrimer structure and the delivery ability into T
cells. The T cell association of these dendrimers was examined at
diﬀerent pHs and temperatures using fluorescence-activated cellsorting (FACS), where murine splenocytes stained with an anti-CD3
antibody were used. Also, we examined the association of PAMAMCHex-Phe and PAMAM-Phe-CHex with some culture cell lines and T
cell subsets, such as CD4-positive helper T cells (CD3+CD4+), CD8positive killer T cells (CD3+CD8+) and activated T cells

2464 | J. Mater. Chem. B, 2022, 10, 2463–2470

Journal of Materials Chemistry B

Fig. 1 Structure of the dendrimers used for the delivery into T cells in thisstudy.

(CD3+CD69+). The intracellular distribution of PAMAM-CHex-Phe
and PAMAM-Phe-CHex was observed using confocal microscopic
imaging. This is the first report on the direct delivery into T cells
using pH-sensitive DDS, as far as we know.

2. Experimental
2.1

Materials

PAMAM-Suc-Phe, PAMAM-CHex-Phe, and PAMAM-Phe-Suc
were synthesized in accordance with our previous reports.5,6
PAMAM-Phe-CHex was synthesized in the same procedure as
for PAMAM-Phe-Suc by replacing Suc with CHex. PAMAMSuc-Leu and PAMAM-CHex-Leu were synthesized using thesame procedures as for PAMAM-Suc-Phe and PAMAM-CHexPhe by replacing Phe with Leu. These carboxy-terminal dendrimers were labeled with fluorescein isothiocyanate (FITC) inaccordance with our previous report.14 The bound number of
FITC to each dendrimer was estimated from the absorbance of
the FITC-conjugated dendrimers at 495 nm. The detailed
synthetic procedures are shown in the ESI.† Phycoerythrin(PE)-conjugated antibody (CD3-PE (clone REA641)) and allophycocyanin (APC)-conjugated antibodies (CD69-APC (clone
REA937), CD4-APC (clone REA604), and CD8a-APC (clone
REA601)) were purchased from Miltenyi Biotec GmbH, Ltd.
(Bergish Gladbach, Germany). Concanavalin A (Con. A) was
purchased from Nacalai Tesque, Inc. (Kyoto, Japan). 4T1 cells
and Jurkat cells were purchased from American Type Culture
Collection (ATCC, VA, USA), and RAW264 cells were obtained
from RIKEN BRC (Ibaraki, Japan). Jurkat cells were cultured ina humidified incubator at 37 1C with 5% CO2 using Roswell
Park Memorial Institute-1640 (RPMI-1640) medium containing
10% of fetal bovine serum (FBS). 4T1 cells and RAW264 cells
were cultured using 10% FBS-containing Dulbecco’s modified
Eagle’s medium (DMEM) and minimum essential medium(MEM), respectively. LysoTracker Red DND-99 and Cellstain

This journal is © The Royal Society of Chemistry 2022

View Article Online

Journal of Materials Chemistry B
DAPI solution were purchased from Thermo Fisher Scientific(Waltham, MA, USA) and Dojindo Laboratories (Kumamoto,Japan), respectively.

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

2.2

Association of dendrimers with T cells

FACS was performed using the same method as in our previous
reports except for the immunostaining and the FACS machine.14
Briefly, spleens were collected from BALB/c mice (7–8-week-old,female) after perfusion with phosphate-buffered saline (PBS) containing heparin under anesthesia. The splenocytes were obtained
after digestion with collagenase for 30 min at 37 1C. Each FITClabeled dendrimer (fluorescent dye concentration: 5 mM) was incubated with 2  105 cells in an RPMI-1640 medium for 3 h at 37 1C or
4 1C with and without pH adjustment to pH 6.5. The cells were
washed and immunostained with CD3-PE, CD4-APC, and CD8a-APC
according to the manufacturer’s instruction. The stained andunstained cells were evaluated by FACS using BD FACS Aria TM
III Cell Sorter (Becton, Dickinson and Company, Franklin Lakes, NJ).
The mean green fluorescence intensity of the singly stained cellswith CD3-PE and double-stained cells with CD3-PE and CD4-APC or
CD8-APC was measured. The cells treated at pH 6.5 were doublestained with CD3-PE and CD69-APC prior to FACS to confirm theT cell activity at pH 6.5.
The splenocytes collected from BALB/c mice were incubated in
10% FBS-containing an RPMI 1640 medium supplemented with
50 mM of 2-mercaptoethanol at a concentration of 3  106 cells per
mL. Then, these cells were cultured in a medium containing Con.
A (1 mg mL 1) for 2 days. Afterward, the FITC-labeled dendrimers
were incubated under diﬀerent conditions, as described above.
These cells were double-stained with CD3-PE and CD69-APC
according to the manufacturer’s instructions. The mean green
fluorescence intensity of the double-stained cells with CD3-PE andCD69-APC (CD3 + CD69+ cells) and the singly stained cells with
CD3-PE (CD3 + CD69- cells) was measured.
The animal experiments were performed in accordance withthe protocols approved by the Animal Care and Use Committees of Osaka Prefecture University.

Paper
2.3

Association of dendrimers with various cell lines

25 mL of RPMI solution containing FITC-labeled dendrimers(fluorescent dye concentration: 5 mM) were added to 1  105
Jurkat cells and incubated at 37 1C or 4 1C for 3 h. Then, 375 mL
of PBS was added to the cell suspension and centrifugated(2000 rpm, 4 1C, 10 min) to collect cells. After washing with
400 mL of PBS, FACS was performed by using GUAVA Incyte,(Luminex, Japan). Adherent RAW264 cells and 4T1 cells wereseeded in a 24-well plate at 1.4  105 cells per well. After
culturing for 24 h, 200 mL of dendrimer solution (fluorescentdye concentration: 5 mM) was added. After the incubation at
37 1C or 4 1C for 3 h, RAW264 cells and 4T1 cells were washedtwice with PBS with and without ethylenediaminetetraacetic
acid (EDTA, 0.05%), respectively. RAW264 cells and 4T1 cells
were detached from the substrate using 200 mL of 0.25% trypsinfor 5 min and 2 min, respectively. A further 200 mL of PBS wasadded to these cell suspensions, and centrifugation was performed (2000 rpm, 4 1C, 10 min) to collect the cells. Afterwashing with 400 mL of PBS, FACS analysis was performed
using GUAVA Incyte. The same procedure was performed byusing dendrimer-containing medium adjusted to pH 6.0 or 6.5
and in the presence of free Phe.

2.4

Confocal microscopic observation

The association of FITC-labeled dendrimers with Jurkat cells
was carried out, as described in 2.3. 75 nM of LysoTracker Red
was added to the cells and incubated at 37 1C. After 1.5 h, the
DAPI stain was added to the medium whose final concentrationof DAPI was 0.7 mg mL 1. After 30 min, cells were collected bycentrifugation (3000 rpm, 4 1C, 5 min). After the resuspensionof cells using serum-containing RPMI medium, cells were
mounted on a glass bottom dish covered with Shi-fix Coverslip(Everest Biotech Ltd, Oxfordshire, UK). After 30 min at 37 1C,
unbound cells were removed by washing with PBS. The cells
were observed using a FV1200 confocal laser scanning microscope (Olympus, Tokyo, Japan).

Fig. 2 Synthetic scheme of the two kinds of PAMAM dendrimers bearing Phe and CHex. (A) PAMAM-CHex-Phe and (B) PAMAM-Phe-CHex. DCC andHBTU are dicyclohexyl carbodiimide and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, respectively, which are condensation agents. TFA is trifluoroacetic acid.

This journal is © The Royal Society of Chemistry 2022

J. Mater. Chem. B, 2022, 10, 2463–2470 | 2465

View Article Online

Paper

Journal of Materials Chemistry B

3. Results

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

3.1 Synthesis and pH sensitivity of the carboxy-terminal
dendrimers bearing Phe
Diﬀerent kinds of hydrophobic carboxy-terminal PAMAM dendrimers were synthesized, as shown in Fig. 1, to compare the
pH-sensitive properties and the association properties of T cells
and their subsets. Previously, PAMAM-CHex-Phe was applied toa delivery into lymph node-resident immune cells.14 PAMAMCHex-Phe was synthesized, as shown in Fig. 2A.5 Anhydride ofCHex was reacted with the PAMAM dendrimer of generation 4(G4) prior to the reaction of benzyl ester of Phe (Phe-OBzl). The
reaction order of Phe and CHex was changed to produce
PAMAM-Phe-CHex (Fig. 2B). Amino-terminal protected Phe(Boc-Phe) was first reacted with the PAMAM dendrimer of
G4.15 After the deprotection by trifluoroacetic acid (TFA), anhydride of CHex was reacted to produce PAMAM-Phe-CHex.
PAMAM-Suc-Phe, PAMAM-CHex-Phe, and PAMAM-Phe-Suc
were already synthesized in our previous reports.5,6 PAMAMSuc-Leu and PAMAM-CHex-Leu, were also synthesized in thesame synthetic procedure by replacing Phe with Leu. The
synthesized dendrimers were characterized by 1H NMR analyses (Fig. S1–S3 (ESI†), respectively). The bound numbers of
Suc and Leu were evaluated using the integral ratios of thesignals at 2.4 ppm and 4.2 ppm, respectively, to that of the
PAMAM signal in the spectra of PAMAM-Suc-Leu (Fig. S1, ESI†).
Since the Suc signal was overlapped with the dendrimer signal,
the integral ratio of Suc was calculated by subtracting that ofthe dendrimer signal. The bound numbers of CHex, Leu, andPhe were evaluated using the integral ratios of the signals at
1.1–1.9 ppm, 0.9 ppm, and 4.2–4.4 ppm, respectively, to those at
2.2–2.4 ppm derived from PAMAM, in the spectra of PAMAMCHex-Leu and PAMAM-Phe-CHex (Fig. S2 and S3, ESI†). The
synthesized dendrimers are listed in Table 1. Almost all theterminal groups of the dendrimer were modified with dicarboxylic acids and amino acids. Each dendrimer was labeledwith a green fluorescent dye, FITC, for the detection of the cellassociated dendrimers. The synthetic procedure was also
shown in our previous report.14 As listed in Table 1, three tonine FITC molecules were conjugated to the dendrimer. The
hydrophobicity indices, log P values, of the terminal groups in
PAMAM-Suc-Phe, PAMAM-Suc-Leu, PAMAM-CHex-Phe, andPAMAM-CHex-Leu were calculated as 0.27, 0.71, 1.2, and0.76, respectively. The log P values of PAMAM-Phe-CHex

Table 1and PAMAM-Phe-Suc were the same as those of PAMAMCHex-Phe and PAMAM-Suc-Phe, respectively (Table 1). Thus,
PAMAM-CHex-Phe and PAMAM-Phe-CHex were the most hydrophobic ones among them.
Our previous reports indicate that PAMAM-Suc-Phe,
PAMAM-CHex-Phe, and PAMAM-Phe-Suc have eﬃcient pHand temperature-responsive properties, but Leu/Ile-conjugateddendrimers do not show any significant stimuli-responsive
properties.4–6 The carboxy-terminal Phe-bearing dendrimers
were soluble under an alkaline condition. However, they were
turbid in acidic pH (Fig. S4, ESI†). PAMAM-Suc-Phe and
PAMAM-Phe-Suc were turbid below pH 4 and pH 5, respectively.
The solution of PAMAM-Phe-Suc, which has both UCST- and
LCST-type thermosensitive properties at different pHs, became
clear below pH 4.7 PAMAM-CHex-Phe and PAMAM-Phe-CHex
were turbid below pH 6 and pH 7, respectively (Fig. S4, ESI†).
Since PAMAM-CHex-Phe and PAMAM-Phe-CHex are morehydrophobic compared with the other ones, the carboxylateions in these dendrimers were protonated at higher pH.6 Also,
the Phe-terminal dendrimers, PAMAM-Suc-Phe and PAMAMCHex-Phe, responded to lower pH than the CHex/Suc-terminal
dendrimers, PAMAM-Phe-Suc, and PAMAM-Phe-CHex. This isbecause the pKa of the carboxy group in Phe is 2.2, which islower than that of carboxylic acid compounds.
3.2 Association of the carboxy-terminal dendrimers bearing
Phe with T cells
The association of the synthesized six kinds of dendrimers withT cells was examined. The splenocytes were collected from miceand incubated with FITC-labeled dendrimers. T cells were
immunostained with CD3-PE, and the association of eachdendrimer with T cells was examined using FACS. The
dendrimer-derived green FITC signal in the CD3-positive(CD3+) cells was examined. Fig. 3 shows that PAMAM-CHexPhe and PAMAM-Phe-CHex were eﬃciently associated withT cells, but the other ones were not. Thus, the combinationof CHex and Phe in the dendrimer is important for the
association with T cells. Selectivity of the cell association is
also important in DDS. Fig. S5 (ESI†) shows the cell distributionof CD3-PE-stained splenocytes, which were associated with
PAMAM-Phe-CHex and PAMAM-CHex-Phe, in the FACS

Summary of carboxy-terminal dendrimers used in this study

Bound number
Dendri-mer

Suc/CHex

Phe/Leu

FITC

log PaTurbidb

PAMAM-Suc-Phe
PAMAM-Suc-Leu
PAMAM-CHex-Phe
PAMAM-CHex-Leu
PAMAM-Phe-Suc
PAMAM-Phe-CHex

64
64
64
64
57
64

64
64
64
64
56
64

7.4
6.7
6.9
9.1
3.2
3.9

0.27
0.71
1.2
0.76
0.27
1.2

pH4
NDc
opH6
NDc
pH5
opH7

a
Calculated log P of the dendrimer terminal group using ChemDraw
software. b Transmittance became less than 20%. c Not determined.

2466 | J. Mater. Chem. B, 2022, 10, 2463–2470

Fig. 3

FACS histograms of the CD3+ T cells at 37 1C.

This journal is © The Royal Society of Chemistry 2022

View Article Online

Journal of Materials Chemistry B

Paper
not (Table 1). These suggested that PAMAM-Phe-CHex was
assembled at around pH 6.5 via hydrophobic interaction.
Enhanced hydrophobicity below pH 7 can promote the T cell
association. Since the microenvironments of tumor tissues are
known to be weakly acidic (pH 6.3–6.9),2 pH-sensitive properties are useful for the selective association with T cells in andaround tumor tissues.
3.3 Association of the carboxy-terminal dendrimers bearing
Phe with Jurkat cells and other cells

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Fig. 4 Mean fluorescence intensity of each dendrimer associated with
CD3+ T cells under diﬀerent conditions. *Higher and p o 0.05 vs. PAMAMCHex-Phe at 4 1C. **Higher and p o 0.05 vs. each normal condition.

analysis. FITC signal was detected from both CD3-PE-stiained
and unstained cells, suggesting that these dendrimers associated with not only T cells but also other cells. The same
experiment was performed at 4 1C, and the mean fluorescenceintensities were compared to those at 37 1C. Fig. 4 shows thatthe fluorescence intensity of all the dendrimers was lower at
4 1C than at 37 1C. This suggests that these dendrimers enter
into cells via endocytosis. The fluorescence intensities of
PAMAM-CHex-Phe and PAMAM-Phe-CHex were relatively higheven at 4 1C compared with the other ones. This suggests thatsome portions of these dendrimers may be adhered to the cellsurface due to hydrophobic interactions. Next, we investigated
the association of each dendrimer with T cells at pH 6.5. The
mean fluorescence intensities of some dendrimers increased at
pH 6.5. Especially, PAMAM-Phe-CHex significantly increased(Fig. 4). The solution containing PAMAM-Phe-CHex became
turbid below pH 7, but that containing PAMAM-CHex-Phe did

Fig. 5

Our results showed that PAMAM-Phe-CHex and PAMAM-CHexPhe were associated with CD3-PE-stained T cells. Since there
are various immune cells including T cells in splenocytes, weused Jurkat cells, human T cell leukemia cells, as a model of T
cells in order to further investigate the association of these
dendrimers with T cells. Fig. S6a (ESI†) shows that PAMAMCHex-Phe was eﬃciently associated with Jurkat cells, but
PAMAM-Phe-CHex was not. The fluorescence intensity was
lower at 4 1C than at 37 1C, which was consistent with theresults using splenocyte-derived T cells. This suggests thatthese dendrimers enter into cells via endocytosis. But, thepH-sensitive behavior was diﬀerent: The mean fluorescenceintensity of PAMAM-CHex-Phe much increased at acidic pH,
but that of PAMAM-Phe-CHex did not. It is possible that theassociation mechanism of these dendrimers with Jurkat cells
may be diﬀerent from murine splenocytes. Confocal microscopic observations were made to confirm the internalizationof these dendrimers into T cells. Fig. 5 shows that both
dendrimers were observed inside Jurkat cells as dotted structures, indicating that both dendrimers were internalized into T
cells via endocytosis. Interestingly, most of these spots did notco-localize with Lysotracker (Fig. S7, ESI†). This suggests that

Confocal microscopic images of Jurkat cells associated with (A) PAMAM-CHex-Phe and (B) PAMAM-Phe-CHex.

This journal is © The Royal Society of Chemistry 2022

J. Mater. Chem. B, 2022, 10, 2463–2470 | 2467

View Article Online

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Paper
these dendrimers possibly inhibited the maturation from endosomes to lysosomes. It was reported that an amino-terminal
PAMAM dendrimer inhibited the maturation to lysosomes,17
consistent with our results. The association of these dendrimers with Jurkat cells was examined in the presence of free Phe
to investigate the eﬀect of Phe to the association. Fig. S8 (ESI†)shows that the dendrimer association slightly increased withincreasing Phe up to 100 mM and then decreased at 200 mM.
These suggest that free Phe has both positive and negativeeﬀects in the association of the dendrimer. The negative eﬀect
is the suppression of Phe-based association of the dendrimer. It
is known that the self-assembled structures of Phe enhance theassociation with the cell membrane and the membrane permeability in PKU.7 Thus, free Phe possibly assist the association of
the dendrimer with cells, which is a possible positive eﬀect.
Further investigation is necessary to elucidate the mechanismof the cellular uptake.
Next, the association of these dendrimers with RAW264 cells(macrophage-like cells) and 4T1 cells (breast tumor cells) was
examined to investigate the cell binding specificity of these
dendrimers to T cells. Fig. S6b and c (ESI†) show that both
dendrimers were also associated with RAW264 cells and 4T1
cells. The fluorescence intensity of these dendrimers was lower
at 4 1C than at 37 1C, suggesting that these dendrimers wereinternalized into these cells via endocytosis like splenocytederived T cells and Jurkat cells. The fluorescence intensity of
PAMAM-Phe-CHex increased at low pH, but PAMAM-CHex-Phe
did not, which was similar to splenocyte-derived T cells.
Because the experimental conditions of these adhesive cells
were diﬀerent from floating splenocytes and Jurkat cells, thedirect comparison of the amount of the cell-associated dendrimer was diﬃcult. Further investigation and improvement are
required for the selective delivery of the dendrimers into T cells.
3.4 Association of PAMAM-Phe-CHex and PAMAM-CHex-Phe
with T cell subsets
Fig. 4 shows that PAMAM-Phe-CHex and PAMAM-CHex-Phe
have the ability of pH-sensitive delivery into T cells. T cells
are classified into CD4-positive (CD4+) helper T cells and CD8positive (CD8+) killer T cells, whose functions are diﬀerent. We
examined the delivery of these dendrimers to these T cellsubsets after double staining with CD3-PE (yellow) and CD4APC or CD8a-APC (red). The green fluorescence signals of eachdendrimer in CD3+CD4+ and CD3+CD8+ cells were examinedusing FACS. Fig. 6 shows that the associations of PAMAM-CHexPhe and PAMAM-Phe-CHex with CD3+CD4+ and CD3+CD8+
cells were similar, indicating that both dendrimers interactedwith both the helper and killer T cells. At 4 1C, the associationsof both dendrimers with these T cell subsets significantly
decreased, indicating that both dendrimers were internalized
into helper and killer T cells via endocytosis. At pH 6.5, theassociations of both dendrimers with the CD3+CD4+ andCD3+CD8+ cells increased. More PAMAM-Phe-CHex was associated with these T cell subsets at pH 6.5 than PAMAM-CHexPhe, which is similar to the results shown in Fig. 4. Thus,
PAMAM-Phe-CHex and PAMAM-CHex-Phe have a delivery

2468 | J. Mater. Chem. B, 2022, 10, 2463–2470

Journal of Materials Chemistry B

Fig. 6 Association of PAMAM-CHex-Phe and PAMAM-Phe-CHex with
CD3+CD4+ helper T cells and CD3+CD8+ killer T cells under diﬀerent
conditions. *Higher and p o 0.05 vs. each normal condition.

potential into helper T cells and killer T cells in a pHsensitive manner, and PAMAM-Phe-CHex is more useful for
pH-sensitive delivery to these T cell subsets in and aroundtumor tissues.
3.5 Association of PAMAM-Phe-CHex and PAMAM-CHex-Phe
with activated T cells
Our results indicated that the associations of both PAMAMCHex-Phe and PAMAM-Phe-CHex with T cells and their subsets
increased at pH 6.5, which is the pH of tumor microenvironments. The function of T cells is known to change under acidic
conditions.9 CD69 is a marker of activated T cells. Con. A is
often used as an activator of T cells to upregulate CD69
expression.16 After the incubation of the splenocytes at
pH 6.5 and/or in the presence of Con. A, they were immunostained with CD3-PE and CD69-APC prior to the FACS analysis.
Fig. S9 (ESI†) shows that the Con. A-untreated T cells did not
express CD69 even at pH 6.5. And, CD69 was expressed in T
cells after the treatment of Con. A. regardless of the adjustment
to pH 6.5. This indicates that T cells are not activated at pH 6.5.
The association of PAMAM-Phe-CHex and PAMAM-CHexPhe with the nonactivated T cells (CD3+CD69-cells) and theactivated T cells (CD3+CD69+ cells) was compared. Fig. 7 showsthat the mean fluorescence intensities of the dendrimers
associated with the activated T cells were higher than thoseof the dendrimers with nonactivated T cells, suggesting thatthese dendrimers can be more eﬃciently associated withactivated T cells than with nonactivated T cells. The fluorescence intensity of PAMAM-Phe-CHex in both the nonactivatedand activated T cells increased at pH 6.5, suggesting that this ismost eﬀectively associated with activated T cells at pH 6.5. Our
results suggest that the association of these dendrimers can becontrolled using pH and T cell activation.

4. Discussion
In this study, we synthesized six carboxy-terminal dendrimers
with hydrophobic amino acids (Phe and Leu) and acidanhydrides (Suc and CHex) in diﬀerent reaction orders toThis journal is © The Royal Society of Chemistry 2022

View Article Online

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Journal of Materials Chemistry B

Fig. 7 Association of PAMAM-CHex-Phe and PAMAM-Phe-CHex with
CD3+CD69+ and CD3+CD69- cells under diﬀerent conditions after theCon. A treatment. *p o 0.05 vs. PAMAM-CHex-Phe or PAMAM-Phe-CHex
under the normal condition in CD3+CD69-cells. **p o 0.05 between thenormal and acidic conditions in CD3 + CD69+ cells.

compare their pH-responsive properties and their association
with T cells and their subsets. The dendrimers bearing CHexand Phe, PAMAM-Phe-CHex and PAMAM-CHex-Phe, showed ahigher association with splenocyte-derived T cells than with theother ones. These dendrimers had the highest log P values inthis study (Table 1), indicating that they are the most hydrophobic ones. This suggests that the hydrophobic eﬀect significantly influences the association of dendrimers with immune
cells. We attempted to elucidate the internalization mechanismof these dendrimers with T cells. As shown in Fig. S8 (ESI†), theassociation of dendrimer with Jurkat cells was slightly
enhanced by the addition of Phe. Because the self-assembled
structure of Phe enhanced the cell membrane permeability,7 itis possible that Phe aggregates at the dendrimer surface directlyinteracted with the cell membranes. The adhesion of thedendrimers to the liposomes as a cell membrane model wasinvestigated (Table S1, ESI†). The adsorption (%) of PAMAMPhe-CHex and PAMAM-CHex-Phe to liposomes was calculated
as 55% and 2%, respectively. This suggested that PAMAM-PheCHex possibly interacted with the cell membranes at thesurfaces of the T cells and their subsets. Fig. S4 (ESI†) showsthat the solubility of PAMAM-Phe-CHex was lower than that of
PAMAM-CHex-Phe even at neutral pH, which supports theresults of the interaction of PAMAM-Phe-CHex with the liposome. Our confocal microscopic analysis indicates that these
dendrimers were internalized into the T cells via endocytosis(Fig. 5). Interestingly, these dendrimers were rarely co-localizedwith lysotracker after the internalization (Fig. S7, ESI†). Further
investigation is necessary.
The targeted delivery is important for DDS. Fig. S5 and S6(ESI†) indicate that our dendrimers were not associated withT cells selectively. Since T cells are classified into various
subsets with diﬀerent functions, the targeted delivery into
specific T cell subsets is also important. Both PAMAM-PheCHex and PAMAM-CHex-Phe were associated with CD4+ helperThis journal is © The Royal Society of Chemistry 2022

Paper
T cells as well as CD8+ killer T cells. These dendrimers showed
pH-responsive properties, as shown in Fig. S4 (ESI†). Especially,
the solubility of PAMAM-Phe-CHex was drastically changed
from a physiological pH of 7.4 to pH 6.5 because of thecoacervation. Although the coacervation did not occur at the
low dendrimer concentration, the dendrimer surface may
become hydrophobic. Thus, the association of PAMAM-PheCHex with T cells and their subsets improved at pH 6.5 (Fig. 4, 6
and 7). Acidic pH is one of the factors of tumor microenvironments. pH-sensitive properties are useful for selective delivery
into cells in and around tumor tissues. Although the acidic pH
did not activate T cells, as shown in Fig. S9 (ESI†), the activatedT cells were more associated with these dendrimers than with
the nonactivated T cells (Fig. 7). The combination of our systemwith T cell activators might also be useful for the selective
delivery to targeted T cells.
We think that our dendrimers are useful for cancer immunotherapy, based on the pH-responsive properties. Activation oflymph node-resident helper T cells and killer T cells as well as
suppression of regulatory T cells localized in tumor tissues arenecessary in cancer immunotherapy. Our previous reports
indicate that anionic terminal dendrimers, including
PAMAM-CHex-Phe, are highly accumulated in the lymph nodeand lymph node-resident T cells.13,14 It is known that small
nanoparticles of less than 70 kDa are necessary for the distribution in the lymph node.18 The molecular weight of ourdendrimers is less than 40 kDa, which is useful for the delivery
into lymph node-resident T cells. This study shows that ourdendrimers have the potential for direct delivery to various
T cells especially at low pH, that is, around tumor cells. Because
dendrimers can load various compounds such as peptides,
drugs, and imaging agents, it is expected that these dendrimers
are a possible carrier for pH-sensitive delivery to metastatic
tumor cell-localized lymph node-resident T cells.

5. Conclusion
In this study, we constructed pH-sensitive delivery systems to
T cells and their subsets by using carboxy-terminal dendrimers
bearing Phe and hydrophobic acid anhydride, such as PAMAMCHex-Phe and PAMAM-Phe-CHex. The dendrimers with Suc
and Leu were not eﬃciently associated with T cells, indicating
the importance of the combination of CHex and Phe for the
association with T cells. These dendrimers were more eﬃciently associated with CD3+ T cells at pH 6.5, which isobserved in tumor microenvironments. This suggests thatthese dendrimers, especially PAMAM-Phe-CHex, are useful fordelivery to T cells around and in tumors. Also, these dendrimers were associated with CD4+ helper T cells and CD8+ killer
T cells at a similar level, suggesting that both cell types are
possible dendrimer targets. These dendrimers were associatedwith the activated CD69+ T cells more eﬃciently. This is thefirst report on the direct delivery into T cells using pH-sensitive
DDS, as far as we know. Our findings are important for thedevelopment of nanoplatforms for direct delivery to T cells to

J. Mater. Chem. B, 2022, 10, 2463–2470 | 2469

View Article Online

Paper
control the functions of T cells, which play key roles in cancer
immunotherapy.

Funding
This work was supported by JSPS KAKENHI Grant Numbers
JP20H05232 (Grant-in-Aid for Scientific Research on Innovative
Area: Aquatic Functional Materials) from the Ministry of Education, Culture, Sports, Science and Technology of Japan(MEXT).

Published on 07 December 2021. Downloaded on 10/8/2024 1:53:38 AM.

Conflicts of interest
H. S. is an employee of Kao Co., Ltd.

Acknowledgements
We are grateful to Prof. Takashi Inui (Osaka Prefecture University) and Dr. Tadahiro Inoue (Osaka Prefecture University)
for their assistance in the animal experiments and for theuseful discussions, respectively.

References
1 S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12,991–1003; H. Wang, Q. Huang, H. Chang, J. Xiao andY.
Cheng,
Biomater.
Sci.,
2016,
4,
375–390;
D. Schmaljohann, Adv. Drug Delivery Rev., 2006, 58,
1655–1670.
2 C. Corbet and O. Feron, Nat. Rev. Cancer, 2017, 17, 577–593;
M. Kanamala, W. R. Wilson, M. Yang, B. D. Palmer andZ. Wu, Biomaterials, 2016, 85, 152–167; T. Haider,
K. K. Sandha, V. Soni and P. N. Gupta, Mater. Sci. Eng., C,
2020, 116, 111229; W. Gao, J. M. Chan and O. C. Farokhzad,
Mol. Pharm., 2010, 7, 1913–1920.
3 D. A. Tomalia, A. M. Naylor and W. A. Goddard, Angew.
Chem., Int. Ed. Engl., 1990, 29, 138–175; S. Svenson and
D. A. Tomalia, Adv. Drug Delivery Rev., 2005, 57, 2106–2129;
C. Kojima, Expert Opin. Drug Delivery, 2010, 7, 307–319.
4 Y. Tono, C. Kojima, Y. Haba, T. Takahashi, A. Harada,
S. Yagi and K. Kono, Langmuir, 2006, 22, 4920–4922.
5 M. Tamaki, D. Fukushima and C. Kojima, RSC Adv., 2018, 8,28147–28151.
6 M. Tamaki and C. Kojima, RSC Adv., 2020, 10, 10452–10460.
7 L. Adler-Abramovich, L. Vaks, O. Carny, D. Trudler,
A. Magno, A. Caflisch, D. Frenkel and E. Gazit, Nat. Chem.
Biol., 2012, 8, 701–706; T. D. Do, W. M. Kincannon and
M. T. Bowers, J. Am. Chem. Soc., 2015, 137, 10080–10083;

2470 | J. Mater. Chem. B, 2022, 10, 2463–2470

Journal of Materials Chemistry B
E. C. Griffith, R. J. Perkins, D. M. Telesford, E. M. Adams,
L. Cwiklik, H. C. Allen, M. Roeselová and V. Vaida, J. Phys.
Chem. B, 2015, 119, 9038–9048; R. Perkins and V. Vaida,
J. Am. Chem. Soc., 2017, 139, 14388–14391; B. G. Anand,
K. Dubey, D. S. Shekhawat and K. Kar, Sci. Rep., 2017, 7, 1–9.
8 I. Mellman, G. Coukos and G. Dranoﬀ, Nature, 2011, 480,
480–489; S. Spranger, H. K. Koblish, B. Horton, P. A. Scherie,
R. Newton and T. F. Gajewski, J. Immunother. Cancer, 2014,
2, 3.
9 V. Hubera, C. Camisaschi, A. Berzi, S. Ferro, L. Lugini,
T. Triulzi, A. Tuccitto, E. Tagliabue, C. Castelli and
L. Rivoltini, Semin. Cancer Biol., 2017, 43, 74–89;
K. Fischer, P. Hoffmann, S. Voelkl, N. Meidenbauer,
J. Ammer and M. Edinger, et al., Blood, 2007, 109,
3812–3819.
10 J. Banchereau and R. M. Steinman, Nature, 1998, 392,
245–252; O. P. Joffre, E. Segura, A. Savina andS. Amigorena, Nat. Rev. Immunol., 2012, 12, 557–569;
M. D. Joshi, W. J. Unger, G. Storm, Y. Kooyk and
E. Mastrobattista, J. Controlled Release, 2012, 161, 25–37.
11 D. Schmid, C. G. Park, C. A. Hartl, N. Subedi,
A. N. Cartwright, R. B. Puerto, Y. Zheng, J. Maiarana,
G. J. Freeman, K. W. Wucherpfennig, D. J. Irvine and
M. S. Goldberg, Nat. Commun., 2017, 8, 1747; S. Cao,S. D. Slack, C. N. Levy, S. M. Hughes, Y. Jiang,
C. Yogodzinski, P. Roychoudhury, K. R. Jerome,
J. T. Schiﬀer, F. Hladik and K. A. Woodrow, Sci. Adv.,
2019, 5, 1–14.
12 L. Tang, Y. Zheng, M. B. Melo, L. Mabardi, A. P. Castaño,Y.-Q. Xie, N. Li, S. B. Kudchodkar, H. C. Wong, E. K. Jeng,
M. V. Maus and D. J. Irvine, Nat. Biotechnol., 2018, 36, 707.
13 Y. Niki, M. Ogawa, R. Makiura, Y. Magata and C. Kojima,
Nanomed. NBM, 2015, 11, 2119–2127; Y. Nishimoto,
S. Nagashima, K. Nakajima, T. Ohira, T. Sato, T. Izawa,
J. Yamate, K. Higashikawa, Y. Kuge, M. Ogawa andC. Kojima, Int. J. Pharm., 2020, 576, 119021.
14 Y. Nishimoto, M. Nishio, S. Nagashima, K. Nakajima,
T. Ohira, S. Nakai, I. Nakase, K. Higashikawa, Y. Kuge,
A. Matsumoto, M. Ogawa and C. Kojima, Polymers, 2020,12, 1474.
15 K. Kono, H. Akiyama, T. Takahashi, T. Takagshi and
A. Harada, Bioconjugate Chem., 2005, 16, 208–214.
16 O. Borges, G. Borchard, A. Sousa, H. E. Junginger and
A. C. Silva, Int. J. Pharm., 2007, 337, 254–264.
17 O. P. Perumal, R. Inapagolla, S. Kannan and R. M. Kannan,
Biomaterials, 2008, 29, 3469–3476.
18 M. A. Swartz and A. W. Lund, Nat. Rev. Cancer, 2012, 12,
210–219.

This journal is © The Royal Society of Chemistry 2022