A Light Responsive Nanoparticle-Based Delivery System Using

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A light responsive nanoparticle-based delivery system using pheophorbide A graft polyethyleneimine for dendritic cell-based cancer immunotherapy Chuangnian Zhang, Ju Zhang, Gaona Shi, Huijuan Song, Shengbin Shi, Xiuyuan Zhang, Pingsheng Huang, Zhihong Wang, Weiwei Wang, Chun Wang, Deling Kong, and Chen Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00015 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Molecular Pharmaceutics

A light responsive nanoparticle-based delivery system using

1 2

pheophorbide A graft polyethyleneimine for dendritic cell-based

3

cancer immunotherapy

4 a, #

, Ju Zhang

b, #

, Gaona Shi a, Huijuan Song a, Shengbin Shi a,

5

Chuangnian Zhang

6

Xiuyuan Zhang a, Pingsheng Huang a, Zhihong Wang a, Weiwei Wang a, Chun Wang a, c

7

*

, Deling Kong a, d *, Chen Li a

8 9

a

Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical

10

Engineering, Chinese Academy of Medical Science & Peking Union Medical College,

11

Tianjin, 300192, China

12

b

13

Shandong Province 26000, China

14

c

15

Hall, 312 Church Street S.E., Minneapolis, MN 55455, USA

16

d

17

Materials, Ministry of Education , Nankai University,300071, China

18 19 20 21 22 23 24 25

Basic Nursing T&R Section, School of Nursing, Qingdao University, Qingdao,

Department of Biomedical Engineering, University of Minnesota, 7-105 Hasselmo

State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive

#

These authors contributed equally to this paper.

Corresponding authors

26 27

Deling Kong, PhD

28

[email protected]

29 30 31 32 33

Chun Wang, PhD [email protected]

ACS Paragon Plus Environment


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1 2

Abstract

3

In this study, the photochemical internalization (PCI) technique was adopted in a

4

nanoparticle-based antigen delivery system to enhance antigen-specific CD8+ T cell

5

immune response for cancer immunotherapy. Pheophorbide A, a hydrophobic

6

photosensitizer, grafted with polyethyleneimine (PheoA-PEI) with endosome escape

7

activity and near infrared imaging capability was prepared. A model antigen

8

ovalbumin (OVA) was then complexed with PheoA-PEI to form PheoA-PEI/OVA

9

nanoparticles (PheoA-PEI/OVA NPs) that are responsive to light. Flow cytometry

10

analysis revealed increased endocytosis in a murine dendritic cell line (DC2.4) that

11

were treated with PheoA-PEI/OVA NPs compared to free OVA. Generation of

12

reactive oxygen species (ROS) in DC2.4 cells was also confirmed quantitatively and

13

qualitatively using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Confocal

14

laser scanning microscopy (CLSM) further demonstrated that the PheoA-PEI/OVA

15

NPs enhanced cytosolic antigen release after light stimulation. Moreover,

16

PheoA-PEI/OVA NPs treated DC2.4 cells exhibited enhanced cross-presentation to

17

B3Z T cell hybridoma in vitro after light irradiation, substantially increased than those

18

treated with free OVA. Consistently, in vivo results revealed upregulation of

19

CD3+CD8+T lymphocytes in tumors of mice treated with dendritic cells plus

20

PheoA-PEI/OVA NPs and light irradiation. The activated T cell response is partly

21

responsible for the inhibitory effect on E.G7 tumor growth in mice immunized with of

22

dendritic cells plus PheoA-PEI/OVA NPs and light irradiation. Our results

23

demonstrate the feasibility to enhance antigen-specific CD8+ T cell immune response

24

by light-responsive nanoparticle-based vaccine delivery for cancer immunotherapy.

25 26

Keywords: photochemical internalization; endosome escape; cross-presentation;

27

antigen delivery; immunotherapy

28 29 30 31


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1 2

1. Introduction

3

Cancer immunotherapy is a treatment to eliminate cancers by activating in vivo

4

cancer-specific immune responses. Because of its high selectivity and low risk of side

5

effects, it is eagerly anticipated as a promising next generation anticancer therapy 1-3.

6

Antigen presenting cells (APCs) such as dendritic cells (DC) and macrophages are

7

known to play a central role in immunity induction. They are also regarded as targets

8

for delivery of tumor-associated antigens to induce cancer specific immune responses

9

4, 5

. Indeed, it has been reported that APCs could induce both antigen-specific humoral

10

and cellular immune responses depending on the routes of delivery of the antigen

11

molecules 6. In general, exogenous antigens such as proteins are taken up by APCs via

12

endocytosis and transferred to the endo/lysosomes where antigens are degraded to

13

peptide fragments and presented by major histocompatibility complexes (MHC) class

14

II molecules. Antigens are thus presented to the CD4+ T cells, which subsequently

15

induce humoral immunity. On the other hand, antigens captured from the extracellular

16

environment by APCs are processed and in the proteasomes and presented on MHC

17

class I molecules to the CD8+ cytotoxic T lymphocytes (CTLs) in a process called

18

“cross-presentation,” resulting in the stimulation of CTLs

19

antigen expressing malignant cells

20

induction and activation of cancer-specific CTLs are crucial because CTL-based

21

cellular immunity directly eliminates the antigen expressing malignant cells. To

22

deliver antigenic molecules into the cytosol of APCs for the induction of

23

antigen-specific cellular immune responses is crucial for effective cancer

24

immunotherapy.

7

and elimination of the

8, 9

. To achieve effective cancer immunotherapy,

25

There are many nanoparticle systems that facilitate antigen delivery into the cytosol

26

and achieve cross-presentation. They also could protect antigen from being degraded

27

by enzymes circulating in the blood and significantly enhance antigen intracellular

28

uptake efficiency through endocytosis

29

that nanoparticles of electrostatically bound PEI and OVA achieved significant

30

antigen cross-presentation by dendritic cells 12. Interestingly, PLL/OVA nanoparticles,

10, 11

. Recently, Xu and colleagues have shown



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1

despite having similar physicochemical properties, were not effective. It was reasoned

2

that endosomal escape of OVA due to the proton sponge effect of PEI was likely

3

responsible for cytosolic delivery and cross-presentation of the antigen. However,

4

dose-dependent cytotoxicity of PEI may limit the applicability of this approach. To

5

further enhance the efficacy of PEI/OVA nanoparticles without potential toxic effect

6

on cells presents a challenge.

7

Photochemical internalization (PCI) is a highly specific, efficient technology with

8

minimal invasiveness to enable cytosolic release of macromolecules entrapped in

9

endocytic vesicles by light

13, 14

. The PCI technology is based on the use of

10

photosensitizers (PS) delivered to endocytic vesicles of cells. The PS is activated

11

upon exposure to light of specific wavelengths, which initiates a photochemical

12

reaction generating reactive oxygen species (ROS, mainly 1O2), resulting in the

13

rupture of endo/lysosomes and cytosolic release of the macromolecules

14

Johansen’s group had reported a series of work on PCI-triggered cytosolic antigen

15

delivery by using a photosensitiser tetraphenyl chlorine disulfonate (TPCS2a). They

16

found that PCI resulted in shift from MHC class II to MHC class I antigen processing

17

and presentation of soluble TPCS2a-OVA complex or liposomes or PLGA

18

microspheres loaded with TPCS2a and OVA for enhanced CD8+ T cell responses and

19

IFN-γ secretion 17-21. Interestingly, Ma et al. found that ROS elicited by nanoparticles

20

could augment proteasome activity and MHC I antigen presentation 22, supporting the

21

fact that PCI may have the potential to enhance CD8+ T cell responses for cancer

22

immunotherapy. However, the majority of existing PS compounds is hydrophobic and

23

easily aggregated under physiological conditions, which drastically lower the

24

quantum yields of ROS production

25

overcome critical limitations of conventional PS drugs. Nanocarriers could enhance

26

the solubility of PS drugs in water through hydrophilic properties and thus increase

27

their cellular uptake

28

(Cdot-Ce6-HA) conjugates, which significantly generated higher singlet oxygen than

29

Ce6 and Cdot-Ce6 in the presence of laser irradiation 25.

30

15, 16

.

23

. Nanomaterials have been employed to

24

. Hahn et al. had prepared carbon dot-chlorine e6-hyaluronate

In this study, we have evaluated the feasibility of a light-responsive


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1

nanoparticle-based antigen delivery system for enhanced antigen-specific CD8+ T cell

2

immune response. The model antigen ovalbumin (OVA) was complexed with a

3

hydrophobic photosensitizer, pheophorbide A (PheoA) grafted polyethyleneimine

4

(PheoA-PEI) to form light responsive nanoparticles (PheoA-PEI/OVA NPs). ROS

5

generation and antigen uptake were assessed in DC2.4 cells. Effects on antigen

6

cross-presentation by the PheoA-PEI/OVA NPs were also investigated in B3Z T cell

7

hybridoma. Importantly, PCI effect on DC based cancer immunotherapy of

8

PheoA-PEI/OVA NPs against E.G7-OVA tumor cells bearing C57BL/6 mice in vivo

9

was investigated.

10 11 12

2. Experimental section

13

2.1. Materials, Cells and Animals

14

Polyethyleneimine (PEI, Mw 10 kDa) was purchased from Aladdin (Shanghai,

15

China). 1-ethyl-3(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC·HCl,

16

Mw 191.70 Da) and N-hydroxysuccinimide (NHS, Mw 115.09 Da) were obtained

17

from GL Biochem Ltd. (Shanghai, China). Pheophorbide-A (PheoA, Mw 592.68 Da)

18

and 9, 10-dimethylanthracene (DMA, Mw 206.28 Da) were obtained from J&K

19

Scientific Ltd. (Beijing, China). Ovalbumin (OVA, Mw 43 kDa) was purchased from

20

Sigma-Aldrich (Beijing, China). Fluorescein isothiocyanate labeled OVA (FITC-OVA)

21

was prepared according to previous report method 26. The cell counting kit-8 (CCK8),

22

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MicroBCA™ Protein

23

assay kit were obtained from Beyotime Biotechnology (Shanghai, China). All the

24

other chemicals were of analytical grade.

25

DC2.4 cells (a murine dendritic cell line) were cultured in RPMI 1640 (Gibco)

26

medium supplemented with 10% fetal bovine serum (Gibco), 100 U/mL

27

penicillin/100 U/mL streptomycin (GIBCO), 2 mM L-glutamine, 55 µM

28

2-mercaptoethanol (Gibco), 1× non-essential amino acids (Cellgro) and 10 mM

29

HEPES (Invitrogen).

30

B3Z Cells were cultured in RPMI 1640 (Gibco) supplemented with 10%



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1

inactivated fetal bovine serum (FBS), 100 U/mL penicillin/100 U/mL streptomycin

2

(Gibco), 2 mM L-glutamine, 55 µM 2-mercaptoethanol (Gibco), and 1 mM sodium

3

pyruvate (Gibco). B3Z is a CD8+ T cell hybridoma specific for MHC-I-restricted

4

SIINFEKL epitope from OVA.

5

E.G7-OVA cells, the murine lymphoma cell line EL4 expressing chicken OVA,

6

were purchased from the American Type Culture Collection (Manassas, VA) and were

7

cultured in RPMI 1640 medium containing 50 µM 2-mercaptoethanol, 10 mM

8

HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin-streptomycin and 10% FBS.

9

All cell lines were grown at 37 oC and 5% CO2.

10

Female C57BL/6 mice purchased from Vital River, Peking, China, were placed in a

11

specific pathogen-free (SPF) environment with a consistent room temperature and

12

humidity. All animal procedures were reviewed and ethically approved by Center of

13

Tianjin Animal Experiment ethics committee and authority for animal protection

14

(Approval No.: SYXK (Jin) 2011-0008).

15 16

2.2. Synthesis and structural characterization of PheoA-PEI

17

PheoA-PEI was synthesized via a conventional carbodiimide reaction. Briefly,

18

EDC·HCl (11.7 mg, 61.0 µmol) and NHS (7.8 mg, 67.8 µmol) were dissolved in 1 mL

19

of DMSO, and the mixtures was mixed with 10 mL of DMSO solution of PheoA (25

20

mg, 42.2 µmol). The activated reaction was carried out for 4 h in the dark at room

21

temperature. The activated PheoA was then added into a DMSO solution of PEI (200

22

mg). The coupling reaction was conducted for 24 h in the dark. The crude reaction

23

mixture was dialyzed against distilled water for 3 days with a molecular weight cutoff

24

of 3.4 kDa. Afterwards, the solution was lyophilized to obtain PheoA-PEI. The

25

coupling ratio for PheoA was characterized using the colorimetric method

26

(Supplemental method 1) 27. The calibration curve of PheoA in DMSO (670 nm) was

27

shown in Figure S2.

28

PheoA-PEI was structurally characterized using proton nuclear magnetic resonance

29

(1H-NMR). 1H-NMR spectra were recorded on an NMR spectrometer (Varian

30

Mercury 400, USA). Fourier-transform infrared (FT-IR) spectroscopy was performed


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1

using an FT-IR spectrometer (Bio-Rad FTS-6000) under ambient conditions.

2

To estimate the fluorescence spectrometric states of PheoA in PheoA-PEI, the

3

PheoA-PEI was dispersed in PBS at a PheoA concentration of 0.5 µg/mL.

4

Fluorescence emission spectra were recorded at 600-750 nm using a fluorescence

5

spectrophotometer (F97 pro, Lengguang Tech, China) with an excitation wavelength

6

of 405 nm.

7 8

2.3. Preparation and Characterization of PheoA-PEI/OVA NPs

9

Both OVA and PheoA-PEI were dissolved in distilled water, which were filtration

10

and sterilization for further use. Nanoparticles were prepared by mixing different

11

mass ratio of OVA (2 mg/mL) and PheoA-PEI (2 mg/mL) by dripping slowly using a

12

vortex for 10 min. Freshly prepared PheoA-PEI/OVA NPs solutions were used in each

13

experiment.

14

The average particle size, polydispersity index (PDI), and zeta potential

15

measurements were made using a Zetasizer 3000 (Malvern Instruments, UK). The

16

morphology of PheoA-PEI/OVA NPs was examined by transmission electron

17

microscopy without staining (TEM, Philips TZOST, Philips Tecnai Co., NED). The

18

near infrared imaging property of PheoA-PEI/OVA NPs were obtained using Maestro

19

imaging system (Maestro, CRI, USA) with a long wave emission filter attached

20

(600-700 nm) and the result was shown in figure S6.

21 22

2.4. Detection of singlet oxygen

23

PheoA-PEI/OVA NPs (1.4 mg) was dispersed in 1 mL Dulbecco’s phosphate

24

buffered saline (DPBS, pH 7.4) and then added to DMA dimethylformamide solution

25

to give a final concentration of 20 µM DMA. One mL of samples containing 140 µg

26

PheoA-PEI/OVA NPs (5 µg/mL of PheoA equivalent) and DMA were irradiated at a

27

light intensity of 5 mW/cm2 using a 670 nm laser (VD-1A, GT2 Laser Power Supply,

28

Beijing Viasho Technology Co., Ltd., China) for different times. The fluorescence

29

spectra of DMA (excitation, 360 nm; emission, 430 nm) as a result of the

30

photosensitization reaction were monitored using a fluorescence spectrophotometer.



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1 2 3

2.5. Determination of intracellular ROS Intracellular

ROS

generation

was

investigated

using

4

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. DCFH-DA is taken up

5

by cells, and then activated by esterase-mediated cleavage of acetate to form

6

dichlorodihydrofluorescein (DCFH), which is trapped in the cells. DCFH is converted

7

to fluorescein DCF in the presence of ROS

8

DCFH-DA in complete medium at 37 oC. After incubation for 30 min in the dark,

9

cells were washed with PBS. PheoA-PEI/OVA NPs (PheoA-PEI/OVA NPs

10

concentration: 140 µg/mL, PheoA concentration: 5 µg/mL) were added and incubated

11

for another 2 h, then 0.5 J/cm2 (5 mW/cm2, 100 s) of irradiation was delivered by a

12

670 nm laser. After that, the cells were harvested and the fluorescence of the cells was

13

detected by a microplate reader to measure the intracellular ROS level. In addition,

14

the intracellular ROS was also detected by observing DCF using confocal laser

15

scanning microscopy using FITC channel (CLSM, TCS SP8, Leica).

28

. DC2.4 cells were incubated with

16 17

2.6. OVA cellular uptake

18

The cellular uptake experiments were performed using flow cytometry and

19

confocal microscopy. For the flow cytometry, immature DC2.4 cells were seeded at

20

5×104 cells per well in 6-well plates for 24 h before treatment. The cells were then

21

treated with FITC-OVA and PheoA-PEI/FITC-OVA NPs (OVA concentration: 50

22

µg/mL). At specific time points, cell medium was removed and cells were washed

23

with PBS, then trypsinized and resuspended in fresh 1640 culture medium. The

24

samples were analyzed by flow cytometry (BD biosciences, San Jose, CA).

25

For confocal microscopy studies, immature DC2.4 cells were seeded in 35 mm2

26

confocal dishes (coverglass-bottom dish) at a density of 1×104 per well for 24 h

27

before treatment. The cells were then treated with PheoA-PEI/FITC-OVA NPs (OVA

28

concentration: 50 µg/mL). One hour later, one group DC2.4 cells were irradiated with

29

a 670 nm laser source, the light intensity was 0.5 J/cm2 (5 mW/cm2, 100 s). After for

30

incubate another one hour, the cell medium was removed and cells were washed with


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1

PBS, followed by soaking in 4% paraformaldehyde for 15 min and washing with

2

deionized water. The cells were imaged with CLSM.

3 4

2.7. Cytotoxicity assay

5

DC2.4 cells were seeded at 1×104 per well into 96-well plates and incubated for 24

6

h. The medium was then replaced with fresh medium containing various

7

concentrations of free OVA and PheoA-PEI/OVA NPs (OVA concentration: 50, 25,

8

12.5, 6.25, 3.125 µg/mL) for 24 h. The cells were irradiated or not with a 670 nm laser,

9

with a power density of 5 mW/cm2 for 100 s. The cytotoxicity of PheoA-PEI/OVA

10

NPs to DC2.4 cells was assessed by using the standard CCK-8 assay. Cells without

11

any treatment were set as control. All the experiments were performed in triplicate.

12 13

2.8. In vitro antigen cross-presentation assay

14

In vitro cross-presentation of OVA by DC was evaluated by a lacZ antigen

15

presentation assay. DC2.4 cells were cultured overnight (5×104 cells/well) in 96-well

16

U-bottom plates. Various concentrations of OVA and PheoA-PEI/OVA NPs (OVA

17

concentration: 50, 25, 12.5, 6.25, 3.125, 1.5625 µg/mL) were added and allowed to

18

incubate for 24 h at 37 °C. H-2Kb-restricted OVA class I epitope SIINFEKL peptide

19

(0.25 µg/mL) and unstimulated DC2.4 cells were used as positive and negative

20

controls, respectively. One group DC2.4 cells incubated with PheoA-PEI/OVA NPs

21

were exposed to light (670 nm) with a power density of 5 mW/cm2 for 100 s. Post

22

incubation, DC2.4 cells were centrifuged (1500 rpm, 5 min) and carefully rinsed

23

twice with 1×DPBS, and B3Z cells (1×105 cells/well) were added and co-cultured

24

with DC2.4 cells for 24 h. Cells were pelleted via centrifugation for 5 min at 1500

25

rpm. The cells were resuspended in 150 µL of CPRG/lysis buffer (1×PBS

26

supplemented with 0.15 mM chlorophenol red-β-D-galactopyranoside, 0.1% Triton-X

27

100, 9 mM MgCl2, 100 µM β-mercaptoethanol) until the color reaction had

28

progressed sufficiently, at which time 100 µL of sample was transferred to 96-well

29

clear flat-bottom plates and the absorbance of released chlorophenol red was

30

measured at 590 nm using a plate reader. All the experiments were performed in



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1

Page 10 of 36

triplicate.

2 3

2.9. Tumor challenge

4

To evaluate antitumor immune response by DC pulsed antigen, DC2.4 cells (1×106)

5

were first incubated with OVA or PheoA-PEI/OVA NPs (OVA concentration: 50

6

µg/mL) for 24 h. Before ending of the incubation time, one group of DC2.4 cells

7

pulsed PheoA-PEI/OVA NPs were exposed to light (670 nm) with a power density of

8

5 mW/cm2 for 100 s. Subsequently, DC2.4 cells were rinsed with warm PBS twice. At

9

the same time, female C57BL/6 6-8 weeks old mice were anesthetized with isoflurane,

10

and 1×106 E.G7-OVA cells were injected subcutaneously into their shaved right flanks.

11

The mice were randomly divided into four groups (n = 6) and vaccinated by

12

transdermal immunization around the tumor site with DC2.4 cells pulsed OVA, DC2.4

13

cells pulsed PheoA-PEI/OVA NPs (without light irradiation) or DC2.4 cells pulsed

14

PheoA-PEI/OVA NPs (with light irradiation) for the first time (1×106 DC2.4 cells in

15

50 µL RPMI 1640). Mice immunized with DC2.4 cells treatment with PBS were used

16

as controls. Each group mice were vaccinated at a one-week interval for four times.

17

Mice were closely monitored every day for tumor growth and body weight. Tumor

18

growth was monitored by recording tumor volume using a digital caliper. Tumor

19

volume (mm3) was calculated as (A×B2)/2, where A and B represent the length and

20

width, respectively. The tumor bearing mice were sacrificed as some of the tumors

21

reached 20 mm in one dimension 29. Tumors were removed and weighted. To study

22

the immune cells in tumors, tumors were harvested from mice in different groups and

23

stained

24

manufacturer’s instructions. Briefly, tumor tissues were cut into small pieces and put

25

into a dish containing RPMI 1640. Then, the single cell suspension was prepared by

26

gentle pressure with the homogenizer. Finally, cells were stained with flow cytometry

27

antibodies after the removal of red blood cells (RBC) using RBC lysis buffer. The

28

percentage of CD3+CD8+ T lymphocytes cells in tumor was assessed by flow

29

cytometry.

30

with

anti-CD3-FITC,

anti-CD8a-PE

antibodies

according

to

the

For in vitro T lymphocytes proliferation assay, splenic T cells were isolated from


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1

C57BL/6 mice of all treatment groups and labeled with 5, 6-carboxyfluorescein

2

acetate N-succinimidyl ester (CFSE, 5 µmol/L, Sigma-Aldrich) according to the

3

manufacturer’s instructions. The CFSE-labeled T cells (4×106 cells/mL) were then

4

incubated with 10 µg/mL soluble OVA antigen for re-stimulation of antigen-specific

5

memory T cells responses and maintained in culture for 5 days. T cells were then

6

collected and percentage of CD8+ CFSElow T cells was assessed by flow cytometry.

7 8

2.10. Statistical analysis

9

The quantitative data collected were expressed as mean ± S.D. Statistical

10

significance was analyzed by student's T-Test. Statistical significance is denoted by *P

11

< 0.05, **P < 0.01 and ***P < 0.001.

12 13 14

3. Results and discussion

15

3.1. Polymer synthesis and structural characterization

16

In the present study, a light-responsive nanoparticulate system composed of

17

pheophorbide-A graft polyethyleneimine and OVA was designed to enhance

18

antigen-specific CD8+ T cell immune response. PheoA, a second generation

19

hydrophobic PS with a longer excitation wavelength, which allows for deeper tissue

20

penetration depth

21

PCI.

30

, was introduced as efficient singlet oxygen photosensitizer for

22

PheoA-PEI was synthesized through a simple one-step carbodiimide coupling

23

reaction between the amino group of PEI and the carboxyl group of PheoA. The

24

synthesis route was shown in Fig. 1A and the chemical structure of PheoA-PEI was

25

verified by

26

(–CH2CH2N– protons) in D2O resonate between 2.2 and 2.8 ppm. The characteristic

27

chemical shifts 3.5-1.1 ppm (1.2, 2.1, 3.1, 3.2, and 3.3 ppm) for CH3, CH, and CH2 in

28

the porphyrin backbone of PheoA were observed in the 1H NMR spectrum of

29

PheoA-PEI (The 1H-NMR of PheoA-PEI in DMSO-d6 was shown in figure S1).

30

Grafting of PheoA onto PEI was further verified using FT-IR (Fig. 1C). Compared

1

H-NMR in D2O (Fig. 1B). The representative signals of PEI



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Page 12 of 36

1

with the PEI IR spectrum, PheoA-PEI gave extra peaks at amide I (1647 cm-1)

2

vibrations, amide II (1387 cm-1) vibrations, indicating that PheoA was successfully

3

linked to PEI. Both 1H NMR and FTIR spectra suggested successful incorporation of

4

PheoA into PEI. Fluorescence spectra of free PheoA, PEI and PheoA-PEI in PBS

5

were shown in Fig. 1D, the fluorescence absorbance peaks of PheoA was at 670 nm,

6

and no difference between PheoA and PheoA-PEI was observable. Content of PheoA

7

in PheoA-PEI conjugate was measured using the colorimetric method, which shows

8

that the PheoA content per 100 mg of PheoA-PEI was 10.7 mg.

9 10 11 12 13

Fig. 1. Synthesis and characterization of PheoA-PEI. (A) Synthesis scheme of PheoA-PEI. (B) H NMR spectra of PEI and PheoA-PEI in D2O. (C) IR spectra of PEI and PheoA-PEI (KBr pellets). (D) Fluorescence spectra of free PheoA in DMSO, PEI and PheoA-PEI in PBS.

14

3.2. Nanoparticle formation and characterization

1

15

Theoretically, PheoA is a hydrophobic PS, so if the number of PheoA in PheoA-PEI

16

is equal, PheoA-PEI could self-assemble into nanoparticles by hydrophobic

17

interactions.

18

polyethyleneimine-glycyrrhetinic acid (PEI-GA) amphiphilic copolymer as a versatile

19

gene/drug dual delivery nanoplatform. They found that PEI-GA with suitable graft

20

ratio of GA could easily self-assemble into nanoaggregates based on the interaction of

21

functional groups among GA, to which DNA could be combined to form PEI-GA

In

previous

studies,

Jiang

and

Zong


et al.

had

prepared

Page 13 of 36

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1

nanoparticles 31. Huh’s group had synthesized PheoA conjugated glycol chitosan (GC)

2

with reducible disulfide bonds (PheoA-ss-GC)

3

aggregation

4

self-assemble in aqueous condition to form core-shell structured nanoparticles with

5

switchable photoactivity for photodynamic therapy. PS in the hydrophobic

6

compartments of various nanoparticles can aggregate and exhibit a self-quenching

7

effect, resulting in the generation of less ROS than in the non-aggregated state 33. In

8

addition, if PheoA-PEI self-assemble into nanoparticles prior to OVA encapsulation,

9

the antigen will be adsorbed on the surface of the nanoparticles, which is less than

10

ideal for antigen protection from enzymatic hydrolysis during in vivo delivery. Hence,

11

electrostatic interaction was adopted to prepare PheoA-PEI/OVA NPs.

concentration,

the

amphiphilic

32

. Upon reaching the critical

PheoA-ss-GC

conjugates

could

The iso-electric point of OVA (43 kDa) is 4.9, and OVA is negatively charged at pH

12

34

13

7

. PheoA-PEI had positive charge at pH 7, so the PheoA-PEI/OVA NPs were

14

simply prepared by the electrostatic interactions of PheoA-PEI and OVA in an

15

aqueous environment. The schematic illustration of formation of PheoA-PEI/OVA

16

NPs and their utility in triggering antigen-specific CD8+ T cell immune response is

17

shown in fig 2A. After PheoA-PEI/OVA NPs were taken up by DC, when light was

18

applied to trigger endosomal escape and cytosolic OVA release, the antigen OVA gets

19

processed and presented by DC and activate CD8+ T cells, which then migrate to the

20

tumor site to attack antigen-specific tumor cells.

21

Different mass ratios of PheoA-PEI and OVA in the preparation of

22

PheoA-PEI/OVA NPs were investigated. The size and zeta potential of

23

PheoA-PEI/OVA NPs were studied and the results are summarized in table S1. As is

24

shown, with the mass of OVA increasing, the size of PheoA-PEI/OVA NPs increased

25

and the zeta potential of PheoA-PEI/OVA NPs was decreased. Then, the dark

26

cytotoxicity assay of PheoA-PEI/OVA NPs against DC2.4 cells was tested. As is

27

shown in supporting information Figure S3, with the mass of PheoA-PEI increasing,

28

the cytotoxicity of PheoA-PEI/OVA NPs increased. Since no evident cytotoxicity

29

could be detected when the mass ratio of PheoA-PEI and OVA was 1: 2, this was then

30

selected for PheoA-PEI/OVA NPs preparation and used in the following study.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Fig 2B is the DLS results of PheoA-PEI, OVA and PheoA-PEI/OVA NPs in water.

2

The average diameter of PheoA-PEI was only about 7 nm. As can be seen, OVA (2

3

mg/mL) aggregated into nano-size with average diameter of approximately 50 nm.

4

Once OVA were added into PheoA-PEI, PheoA-PEI/OVA NPs were formed by the

5

electrostatic reaction. The average diameter of PheoA-PEI/OVA NPs changed to 276

6

nm. These results demonstrated that the PheoA dispersed within the PheoA-PEI/OVA

7

NPs, which is beneficial for the generation of ROS and near infrared imaging, as

8

incorporated into polymeric nanocarrier systems easily form aggregates due to

9

hydrophobic characteristics, resulting in significant reduction of singlet oxygen by 35

10

self-photo quenching

. TEM revealed that PheoA-PEI/OVA NPs were spherical

11

structures with an average diameter between 100 and 200 nm (fig 2C), smaller than

12

the hydrodynamic diameter obtained from the DLS experiment.


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1 2 3 4 5 6 7

Fig. 2. Preparation and characterization of PheoA-PEI/OVA NPs. The nanoparticles concentration is 2 mg/mL. (A) Schematic representation of the formation of PheoA-PEI/OVA NPs and their utility in triggering antigen-specific CD8+ T cell immune response. (B) DLS measurement of PheoA-PEI, OVA and PheoA-PEI/OVA NPs. (C) TEM image of PheoA-PEI/OVA NPs.

8

3.3. ROS detection

9

PheoA was selected as PS in photodynamic therapy in previous study

32, 36

. In this

10

study, PheoA-PEI was not only used as the carrier to form nanoparticles, but also as a

11

highly efficient photosensitizer to generate ROS in PCI. The ability of A-PEI/OVA



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

NPs to generate ROS under light irradiation is crucial and was assessed by the

2

common DMA bleaching methods to measure singlet oxygen released in the medium.

3

Fluorescence intensities of DMA were used for quantification of singlet oxygen

4

generation by PheoA-PEI/OVA NPs. It is known that DMA reacts irreversibly with

5

1

6

DMA absorption band at 360 nm. Fig. 3A, B shows the fluorescence activity of DMA

7

co-incubated with PheoA-PEI/OVA NPs with 670 nm light irradiation (5 mW/cm2) at

8

different time periods. Control experiments in the absence of PheoA-PEI/OVA NPs

9

revealed that DMA was stable during irradiation (data not shown). Our results showed

10

that the intensity of PheoA-PEI/OVA NPs after 6 min exposure decreased

11

substantially, indicating rapid generation of singlet oxygen upon light irradiation.

O2 in many organic solvents and water, and causes a decrease in the intensity of the

12

As the level of intracellular ROS is an important indicator for PCI, intracellular

13

ROS generation was investigated quantitatively and qualitatively using a DCFH-DA

14

assay. Briefly, DCFH-DA is taken up by cells, and activated by esterase-mediated

15

cleavage of acetate to form dichlorodihydrofluorescein (DCFH), which is trapped

16

within the cells, and convert to fluorescein DCF in the presence of ROS. Fig. 3C

17

shows the mean fluorescence intensity of DC2.4 cells incubated with DCFH-DA

18

probe in medium with and without PheoA-PEI/OVA NPs. As can be seen, the mean

19

fluorescence intensity of DC2.4 cells with PheoA-PEI/OVA NPs under light

20

irradiation was 3 times of that without light irradiation. Fig. 3D shows the CLSM

21

images of illuminated DC2.4 cells incubated with DCFH-DA probe in media with and

22

without PheoA-PEI/OVA NPs. The green fluorescence signal was almost undetectable

23

in DC2.4 cells cultured alone and DC2.4 cells treated PheoA-PEI/OVA NPs without

24

light irradiation. In contrast, after being treated with PheoA-PEI/OVA NPs, DC2.4

25

cells were exposed to light irradiation, and strong fluorescence signals that evenly

26

distributed throughout the cells could be observed, indicating that PheoA-PEI/OVA

27

NPs could generate ROS in DC2.4 cells under 670 nm light irradiation and had the

28

potential to be used in PCI mediated antigen cytoplasm delivery.


Page 16 of 36

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1 2 3 4 5 6 7 8

Fig. 3. Singlet oxygen generation. (A) Fluorescence emission spectra of PheoA-PEI/OVA NPs with DMA solution with an increase in the light irradiation time. (B) The fluorescence intensity change of DMA at 432 nm as a function of light irradiation time. (C) Microplate reader analysis and (D) CLSM images of intracellular ROS generation determined by measuring the fluorescence intensity of DCF in DC2.4 cells. (Light intensity was 0.5 J/cm2 using a 670 nm laser source)

9

3.4. Cytotoxicity assay

10

PheoA had been used in PDT for cancer therapy in previous studies. The aim of

11

PDT was to deliver PheoA to tumor cells, generate ROS by light irradiation and kill

12

tumor cells. In this work, both PS and antigen were delivered to DC2.4 cells

13

simultaneously. ROS produced by PheoA was utilized to disrupt the endosome and

14

facilitate cytoplasmic antigen exposure without harming the DC, so it is necessary to

15

assess DC survival rate. The cytotoxicity of PheoA-PEI/OVA NPs was tested in

16

DC2.4 cells using CCK-8 cell viability assay by exposing DC2.4 cells to a series of

17

concentrations up to 50 µg/mL, the concentration used for subsequent MHC-I

18

presentation assay. The light intensity used in this work was reference for Na’s work

19

37

20

with untreated cells. It can be seen in Fig. 4 that none of PheoA-PEI/OVA NPs induce

21

toxicity in DC2.4 cells at this concentration regardless of exposure of light irradiation.

. Cell survival was calculated by comparing the PheoA-PEI/OVA NPs treated cells



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4

Fig. 4. DC2.4 Cells viability treated with various concentrations of free OVA and PheoA-PEI/OVA NPs with or without light irradiation. (Light intensity was 0.5 J/cm2 using a 670 nm laser source)

5 6

3.5. Cell uptake

7

One of the major advantages of nanoparticle based antigen delivery system is

8

enhanced intracellular antigen uptake, which could potentially result in increased

9

antigen cross-presentation and subsequent CTL reaction.

10

To quantify the cellular uptake of free OVA and PheoA-PEI/OVA NPs, FITC

11

labeled OVA was used. FITC-OVA loaded nanoparticles were prepared without

12

modification and no detectable changes were observed regarding the size and zeta

13

potential of FITC-OVA compared to unlabeled OVA. The fluorescence intensity in

14

DC2.4 cells after incubation with FITC-OVA and PheoA-PEI/OVA NPs was measured

15

by flow cytometry. Fig. 5A shows the flow cytometry histograms of DC2.4 cells after

16

incubation with FITC-OVA and PheoA-PEI/OVA NPs at 2 h and 24 h. The mean

17

fluorescence intensities of DC2.4 cells for FITC-OVA and PheoA-PEI/OVA NPs were

18

75878.3 and 234383.9 at 2 h, respectively, indicating approximately a 2.8-fold

19

improvement in cell uptake of PheoA-PEI/OVA NPs. As time extended, the mean

20

fluorescence intensity of DC 2.4 cells did not change in the FITC-OVA group,

21

whereas mean fluorescence intensity of DC2.4 cells of the PheoA-PEI/OVA NPs

22

group increased to 325584.0, indicating increased antigen uptake by DC2.4 cells from

23

the NP group.

24

To stimulate strong CD8+ T-cell responses, cytoplasmic antigen release is necessary.


Page 18 of 36

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1

The intracellular localization of OVA and PheoA-PEI/OVA NPs within DC2.4 cells

2

was examined using confocal laser scanning microscopy (CLSM). LysoTracker Red,

3

a fluorescence probe was employed to examine whether the PheoA-PEI/OVA NPs

4

were localized in endosomes and lysosomes. As shown in Fig. 5C, DC2.4 cells after

5

treated PheoA-PEI/OVA NPs without light irradiation, most of the green fluorescence

6

signals of FITC -OVA in DC2.4 cells was co-localized with lysoTracker (as indicated

7

by orange). In contrast, after light irritation, the green fluorescence signals from the

8

FITC-OVA displayed a more diffused pattern in the cytoplasm. Moreover, possible

9

endosomal disruption could be depicted as decreased red fluorescence intensity of the

10

LysoTracker after light irradiation. In previous studies, one common method for

11

endosomal escape was the pH-buffering effect (the proton sponge effect)

12

pH-buffering effect of PEI could lead to an increase in osmotic pressure in the

13

endosome that result in disruption of the endosomal membrane. However, in our study,

14

little endosomal escape was observed in DC2.4 cells treated with PheoA-PEI/OVA

15

NPs without light irradiation. Our results suggest that PheoA-PEI/OVA NPs had the

16

ability to deliver antigen to the cytosol in DC2.4 cells by PCI-mediated endosomal

17

disruption.


38

. The


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10

Fig. 5. Cellular uptake and endosomal release of nanoparticles by DC2.4 cells. (A) Flow cytometry assay results for the cellular uptake of free OVA and PheoA-PEI/OVA NPs at 2 h and 24 h. (B) The intracellular mean fluorescence intensity of DC2.4 cells after incubate with free OVA and PheoA-PEI/OVA NPs. The fluorescence of cells was analyzed by a Becton-Dickinson flow cytometer with 488 nm excitation (C) Confocal microscope images of DC2.4 cells after incubate with PheoA-PEI/OVA NPs with or without light irradiation. The bar scale indicates 7.5 µm. (Light intensity was 0.5 J/cm2 using a 670 nm laser source)

3.6. In Vitro MHC-I Antigen Presentation

11

Cross-presentation of endocytosed tumor-associated antigens to cytotoxic CD8+

12

T-cells is essential for the induction of antitumor immunity. The ability of polymeric

13

nanoparticles to enhance MHC class I antigen presentation was assessed by an in vitro

14

antigen presentation assay using DC2.4 cells as the antigen presenting cell and a

15

specialized LacZ B3Z T cell hybridoma that produce β-galactosidase upon

16

recognition of the immunodominant ovalbumin class I epitope SIINFEKL presented

17

on MHC class I H-2Kb on DC2.4 cells. Expression of β-galactosidase can be

18

quantified by addition of CPRG, a substrate for the β-galactosidase, as quantification


Page 20 of 36

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1

of antigen cross-presentation. As shown in Fig. 6, at specified concentrations, the

2

MHC-I antigen presentation in the OVA-only group was very low, in contrast to the

3

PheoA-PEI/OVA NPs group which resulted in significant increase of MHC-I antigen

4

presentation regardless of light irradiation. This result indicated that nanoparticle

5

based antigen delivery system is superior than the antigen alone in activation MHC

6

class I cross-presentation. Three possible mechanisms may be deduced from the study,

7

one of which is enhanced antigen uptake in DC2.4 cells by PheoA-PEI/OVA NPs.

8

Another explanation is DC2.4 cells treated with PheoA-PEI/OVA NPs under light

9

irradiation (50 µg/mL OVA) for 24h showed a much higher expression of surface

10

CD40, CD80, and CD86 than those cultured with OVA or PheoA-PEI/OVA NPs

11

without light irradiation, indicating DC2.4 cells maturation (Figure S5). The last one

12

is that parts of antigen OVA may escape from the endosomes during the

13

PheoA-PEI/OVA NPs delivery due to the proton sponge effect of PEI. Upon light

14

irradiation, the OD had further improved, indicating ROS had play great role in the

15

MHC-I antigen presentation.

16 17 18 19 20

Fig. 6. In vitro protein cross-presentation. DC2.4 cells were incubated with free OVA or PheoA-PEI/OVA NPs with or without light irradiation and subsequently co-cultured with B3Z T cells which produce β-galactosidase in response to antigen presentation on MHC-I. (Light intensity was 0.5 J/cm2 using a 670 nm laser source)

21 22 23 24

3.7. Tumor challenge Treatment with DC pulsed with disease associated antigens has been previously reported as effective in suppressing tumor growth

39

. To investigate whether



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

PheoA-PEI/OVA NPs can be used in DC based cancer immunotherapy, we evaluated

2

the therapeutic performance using mice bearing E.G7-OVA tumors which are

3

responsive to antigen OVA. Immediately after inoculation with E.G7-OVA tumor cells,

4

we immunized the E.G7-OVA tumor-bearing C57BL/6 mice 3 times with 1 week

5

apart with DC2.4 cells plus antigen. Mice received only DC2.4 cells were used as

6

control. As shown in Fig. 7B, both DC2.4 cells plus PheoA-PEI/OVA NPs-treated

7

group (with light) and DC2.4 cells plus PheoA-PEI/OVA NPs-treated group (without

8

light) exhibited significantly inhibited tumor growth compared to DC2.4 cells - and

9

DC2.4 cells plus OVA-treated groups. Consistently, reduced mean tumor volumes

10

were also observed by day 28 in DC2.4 cells plus PheoA-PEI/OVA NPs with light

11

irradiation (133.69 mm3) and DC2.4 cells plus PheoA-PEI/OVA NPs without light

12

irradiation (426.99 mm3), significantly less than the DC2.4 cells only control group

13

(2696.64 mm3) and DC2.4 cells plus OVA group 2386.49 mm3). Similarly, mean

14

tumor weights of mice treated with DC2.4 cells plus PheoA-PEI/OVA NPs (with light

15

irradiations), DC2.4 cells plus PheoA-PEI/OVA NPs (without light irradiations),

16

DC2.4 cells plus OVA and DC2.4 cells alone on day 28 were 0.05 g, 0.28 g, 2.35 g

17

and 2.52 g, respectively (Fig. 7C). The photographs of tumor blocks isolated on day

18

28 further confirmed obvious shrinkage of tumors from the DC2.4 cells plus

19

PheoA-PEI/OVA NPs groups (Fig. 7D). No obvious body weight loss or noticeable

20

abnormality could be detected in groups received PheoA-PEI/OVA NPs regardless of

21

light irradiations, indicating no onset of acute toxicity of the nanoparticle formulations

22

or light irradiations. In contrast, evident weight gain could be observed in the DC2.4

23

cells- and DC2.4 cells plus OVA-treated groups and the increase of body weight is

24

due to rapid tumor growth (Fig. 7A).

25

The therapeutic response of mice immunized with antigen was mainly dependent

26

on the presence and activities of CD8+ T cells 40. As shown in Fig. 7E, among tumor

27

infiltrating cells, there is no obvious difference in the percentage of CD3+CD8+ T

28

lymphocytes between the control group (2.37%) and DC2.4 cells plus OVA-group

29

(2.33%). However, we detected significantly increased infiltration of CD3+CD8+ T

30

lymphocytes within tumors obtained from mice immunized with DC2.4 cells plus


Page 22 of 36

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1

PheoA-PEI/OVA NPs (6.34% without light irradiations vs. 12.78% with light

2

irradiations).

3

T-cell proliferation is another key immunological event following immune

4

activation 41, which could be mearused by CFSE labeling of CD8+ T cells. As shown

5

in Fig. 7F, no differences of CD8+CFSElow T cell proliferation were detectable

6

between the control group (8.21%) and DC2.4 cells plus OVA group (8.78%).

7

However, CD8+ T cells proliferation increased to 11.41% and 13.04% in mice treated

8

with DC2.4 cells plus PheoA-PEI/OVA NPs without light irradiations and with light

9

irradiations, respectively, demonstrative of robust ability of PheoA-PEI/OVA NPs on

10

CD8+ T cell activation and proliferation, both of which are responsible for the

11

substantial tumor regression.

12



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1 2 3 4 5 6 7 8

Fig. 7. In vivo antitumor immune response in E.G7-OVA tumor-bearing C57 mice. (A) The body weight, (B) tumor volume and (C) tumor weight of mice in different treatment groups. (D) The photographs of typical tumor blocks collected from different treatment groups of mice on day 28. (E) Phenotype analysis of CD3+CD8+ T cells in tumor. (F) CFSE labeled CD8+ T cell proliferation (defined as CFSElow) in spleen. Data are presented as mean ± SD (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test).

9

4. Conclusions

10

In this study, we have shown the therapeutic effectiveness of light responsive

11

nanoparticles (PheoA-PEI/OVA NPs) as antigen delivery system for cancer

12

immunotherapy. Enhanced antigen uptake was observed by PheoA-PEI/OVA NPs in

13

DC2.4 cells. Upon light irradiation, PheoA-PEI/OVA NPs could promote intracellular

14

ROS generation, which effectively promote antigen endosomal escape, leading to

15

cytosolic antigen release. Importantly, compared with free OVA, PheoA-PEI/OVA

16

NPs treated DC2.4 cells showed enhanced cross-presentation to B3Z T cell

17

hybridoma in vitro after light irradiation. Moreover, the incorporation of DC2.4 cells

18

plus PheoA-PEI/OVA NPs with light irradiation drastically enhances CTL activities

19

and antitumor effects in vivo. In summary, our results showed that the

20

PheoA-PEI/OVA NPs have great potential as a vaccine delivery system that could

21

induce antigen-specific cellular immunity for cancer immunotherapy.

22 23 24

Acknowledgments

25

This work was financially supported by the National Natural Science Foundation of

26

China (21604095, 31300732, 51373199, 31670977), China Postdoctoral Science

27

Foundation (2015M580066)，Program for Innovative Research Team in Peking Union

28

Medical College, CAMS Initiative for Innovative Medicine (2016-I2M-3-022) and the

29

Open Fund of Key Laboratory of Functional Polymer Materials, Ministry of

30

Education, Nankai University (201606).

31 32


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1

This document file contains Supporting Information.

2

1. Determination of PheoA content in PheoA-PEI.

3

2. DC maturation measurement.

4

Table S1. Characterization of PheoA-PEI/OVA NPs.

5

Figure S1. 1H NMR analysis of PheoA-PEI in DMSO-d6.

6

Figure S2. The calibration curve of PheoA in DMSO(670 nm).

7

Figure S3. The DC2.4 Cells viability treated with various concentrations of free OVA and three

8

kinds of PheoA-PEI/OVA NPs with different mass ratio.

9

Figure S4. Diameter change of PheoA-PEI/OVA NPs in water.

10

Figure S5. DC maturation induced by OVA and PheoA-PEI/OVA NPs in vitro.

11

Figure S6. The near infrared image of PheoA-PEI/OVA NPs in water.

12 13 14

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1

cytotoxic T lymphocytes. Biomaterials 2016, 77, 243-254.


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Fig. 1. Synthesis and characterization of PheoA-PEI. (A) Synthesis scheme of PheoA-PEI. (B) 1H NMR spectra of PEI and PheoA-PEI in D2O. (C) IR spectra of PEI and PheoA-PEI (KBr pellets). (D) Fluorescence spectra of free PheoA in DMSO, PEI and PheoA-PEI in PBS. 160x92mm (300 x 300 DPI)



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Fig. 2. Preparation and characterization of PheoA-PEI/OVA NPs. The nanoparticles concentration is 2 mg/mL. (A) Schematic representation of the formation of PheoA-PEI/OVA NPs and their utility in triggering antigenspecific CD8+ T cell immune response. (B) DLS measurement of PheoA-PEI, OVA and PheoA-PEI/OVA NPs. (C) TEM image of PheoA-PEI/OVA NPs. 160x215mm (300 x 300 DPI)


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Fig. 3. Singlet oxygen generation. (A) Fluorescence emission spectra of PheoA-PEI/OVA NPs with DMA solution with an increase in the light irradiation time. (B) The fluorescence intensity change of DMA at 432 nm as a function of light irradiation time. (C) Microplate reader analysis and (D) CLSM images of intracellular ROS generation determined by measuring the fluorescence intensity of DCF in DC2.4 cells. (Light intensity was 0.5 J/cm2 using a 670 nm laser source) 160x95mm (300 x 300 DPI)



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Fig. 4. DC2.4 Cells viability treated with various concentrations of free OVA and PheoA-PEI/OVA NPs with or without light irradiation. (Light intensity was 0.5 J/cm2 using a 670 nm laser source) 80x61mm (300 x 300 DPI)


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Fig. 5. Cellular uptake and endosomal release of nanoparticles by DC2.4 cells. (A) Flow cytometry assay results for the cellular uptake of free OVA and PheoA-PEI/OVA NPs at 2 h and 24 h. (B) The intracellular mean fluorescence intensity of DC2.4 cells after incubate with free OVA and PheoA-PEI/OVA NPs. The fluorescence of cells was analyzed by a Becton-Dickinson flow cytometer with 488 nm excitation (C) Confocal microscope images of DC2.4 cells after incubate with PheoA-PEI/OVA NPs with or without light irradiation. The bar scale indicates 7.5 µm. (Light intensity was 0.5 J/cm2 using a 670 nm laser source) 160x137mm (300 x 300 DPI)



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Fig. 6. In vitro protein cross-presentation. DC2.4 cells were incubated with free OVA or PheoA-PEI/OVA NPs with or without light irradiation and subsequently co-cultured with B3Z T cells which produce β-galactosidase in response to antigen presentation on MHC-I. (Light intensity was 0.5 J/cm2 using a 670 nm laser source) 80x60mm (300 x 300 DPI)


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Fig. 7. In vivo antitumor immune response in E.G7-OVA tumor-bearing C57 mice. (A) The body weight, (B) tumor volume and (C) tumor weight of mice in different treatment groups. (D) The photographs of typical tumor blocks collected from different treatment groups of mice on day 28. (E) Phenotype analysis of CD3+CD8+ T cells in tumor. (F) CFSE labeled CD8+ T cell proliferation (defined as CFSElow) in spleen. *p < 0.05, **p < 0.01, and ***p < 0.001 (Student's t test). 160x165mm (300 x 300 DPI)



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44x31mm (300 x 300 DPI)


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A Light Responsive Nanoparticle-Based Delivery System Using

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