Biomimetic polymeric nanoparticle-based photodynamic immunotherapy and protection against tumor rechallenge

Dongyoon Kim , Junho Byun , Jinwon Park , Yeon Lee , Gayong Shim * and Yu-Kyoung Oh *
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. E-mail:;

Received 23rd October 2019 , Accepted 9th January 2020

First published on 10th January 2020

In this study, we sought to design a bionanomaterial that could exert anticancer effects against primary tumors and protect against rechallenged tumors via photodynamic immunotherapy. As a biomaterial, we used an amphiphilic phenylalanine derivative of poly-gamma glutamic acid, which forms nanoparticles by self-assembly. For anticancer effects, we co-entrapped hydrophobic chlorin e6 and monophosphoryl lipid A in the core of the plain amphiphilic phenylalanine nanoparticles (AN), to generate M/C/AN. For comparison, we used plain AN and chlorin e6-loaded AN (C/AN). In vitro studies showed that B16F10 cancer cells treated with C/AN or M/C/AN generated reactive oxygen species and exhibited an enhanced surface display of calreticulin upon exposure to 660 nm light irradiation. C/AN and M/C/AN exerted similar photodynamic anticancer effects; however, M/C/AN, but not C/AN, induced in vitro dendritic cell maturation. Our biodistribution study revealed that C/AN and M/C/AN showed higher accumulation at the tumor tissues compared to that seen in the free chlorin e6-treated group. In B16F10 tumor-bearing mice, the intravenous injection of C/AN or M/C/AN showed similar photodynamic anticancer effects against primary tumors. However, the growth of rechallenged tumors was more significantly inhibited in the M/C/AN group compared to the C/AN group. At day 40 after inoculation of the primary tumor, M/C/AN-treated mice showed 100% survival, whereas the other groups showed 0% survival. In the tumor microenvironment, higher infiltration of CD8+ T cells was observed in the M/C/AN group compared to the other groups. Our results suggest that AN co-loaded with a photosensitizer and an immune stimulant may hold great potential for use in photodynamic immunotherapy to inhibit both primary and metastatic tumors.


Photodynamic therapy (PDT) has been used as a noninvasive modality for cancer treatment. In PDT, light is used to cause photosensitizer molecules to generate reactive oxygen species (ROS) that kill cancer cells.1 PDT lacks the side effects of anticancer chemotherapeutics and does not require surgery, and is currently in clinical use for the treatment of superficial tumors, such as skin and gynecological cancers.2,3

Because the photosensitizers identified to date are hydrophobic and show poor tumor accumulation, researchers have sought to deliver them using various nanomaterials.4,5 For example, conjugation of a hydrophobic photosensitizer to poly(glycidyl methacrylate) was shown to improve the anticancer efficacy of PDT, compared to the use of an unconjugated hydrophobic photosensitizer.4,6 In addition, a cyclodextrin-based polycation derivative has been used to encapsulate a photosensitizer in a hydrophobic core.5

Despite the noninvasive character of PDT, its current application is limited to the treatment of primary cancers.7 Thus, if even a few cancer cells have metastasized, the current PDT strategies will fail to eradicate cancer. In clinical situations, patients may undergo regular medical check-ups and other treatment modalities aimed at preventing metastasis after PDT. Thus, it would be desirable to develop a PDT-based strategy that can exert anticancer effects against both primary and metastatic tumors.8

Recently, PDT was shown to enhance immunogenic cell death by activating anti-tumor immunity.9 Although immunotherapy has emerged as an attractive way to combat metastasized cancer cells, immunotherapy alone may not be sufficient to ablate tumors. These efforts may require the combination of immunotherapy with other treatment modalities, such as chemotherapy and radiation therapies. To minimize the side effects of chemotherapy and radiation and to enhance the potency of immunotherapy, we need a new treatment modality that can be combined with immunotherapy.

The PDT-triggered generation of ROS is known to induce danger associated molecular patterns (DAMP) such as calreticulin (known as an “eat me” signal).10 The resulting DAMP-mediated uptake of ROS-damaged tumor cells by dendritic cells (DCs) can promote the maturation of DCs. Mature DCs reportedly present tumor antigens and prime T cells.11 Thus, the induction of DAMP with external light stimuli would be an effective way to increase the potency of immunotherapy.

In this study, we sought to design a nanoparticle that can provide anticancer effects against primary and distant tumors (Fig. 1). To achieve this, we co-entrapped chlorin e6 (Ce6) as a photosensitizer and monophosphoryl lipid A (MPL) as an immune stimulant in nanoparticles made of an amphiphilic phenylalanine derivative of poly-gamma glutamic acid (PGA). Ce6 is a well-known porphyrin-like photosensitizer in PDT, and 660 nm irradiation of Ce6 generates ROS, including singlet oxygen (1O2) and hydroxyl radicals (˙OH).12,13 PGA is the main component of the biofilm generated by certain bacteria that are able to avoid interacting with blood cells (e.g., Bacillus anthracis).14 Here, we report that the co-entrapment of Ce6 and MPL (an immune stimulator of Toll-like receptor 4) yielded a nanoparticle that exerts anticancer effects against both primary and rechallenged tumors.

image file: c9bm01704f-f1.tif
Fig. 1 Schematic illustration of M/C/AN-mediated photodynamic immunotherapy. PGA was conjugated with a hydrophobic moiety, and the generated AP nanoparticles were utilized for co-encapsulation of Ce6 and MPL (A). Tumor-bearing mice were systemically injected with M/C/AN nanoparticles and, at 1 day after injection, the tumor sites were irradiated with red light at 660 nm for PDT (B). The M/C/AN-treated and light-irradiated cancer cells act as an effective in situ cancer vaccine due to PDT-mediated DAMP signaling and the action of MPL.


Preparation of nanoparticles

AP was synthesized as described previously.15 Briefly, the carboxyl groups of PGA (MW, 50 kDa; Bioleaders Corp., Daejeon, Republic of Korea) were conjugated with the amino group of phenylalanine ethyl ester (L-phenylalanine ethyl ester hydrochloride; Sigma-Aldrich, St Louis, MO, USA). The M/C/AN nanoparticles were prepared using the thin-film hydration method.16 To prepare plain AP nanoparticles (AN), 5 mg of AP, 100 μg of Ce6 (Santa Cruz Biotechnology, Dallas, TX, USA) and 100 μg of MPL (InvivoGen, San Diego, CA, USA) were dissolved in 1 mL of methanol. To prepare Ce6-loaded AN(C/AN), 100 μg of Ce6 was dissolved in 1 mL of methanol. To prepare AN co-loaded with MPL and Ce6 (M/C/AN), 100 μg of Ce6 and 100 μg of MPL were dissolved together in 1 mL of methanol. In each case, the solvent was removed and the resulting film was hydrated with 1 mL of phosphate-buffered saline (PBS) by vigorous vortexing and sonication for 30 min. For C/AN or M/C/AN, the unloaded compounds were removed with a PD SpinTrap G-25 (GE Healthcare, Buckinghamshire, UK).

Characterization of nanoparticles

The sizes and zeta potentials of nanoparticles were measured by dynamic light scattering using a He–Ne laser and laser Doppler microelectrophoresis at an angle of 22°, respectively (ELSZ-1000; Otsuka Electronics Co., Osaka, Japan). The morphologies of the nanoparticles were assessed by transmission electron microscopy (TEM) (Talos L120C; Thermo Fisher Scientific, Inc., Waltham, MA, USA). For TEM imaging, nanoparticles were prepared on a 12 mm-diameter copper grid (FESEM finder grid; Ted Pella Inc., Redding, CA, USA).

Drug-loading test

The loading amounts of Ce6 and MPL were measured by UV-Vis spectrophotometry and the phosphate-quantifying Fiske–Subbarow method, respectively.17 The contents of Ce6 were analyzed by assessing absorbance at 401 nm using a SpectraMAX M5 (Molecular Devices, San Jose, CA, USA) with standard-curve calibration. To assay the loading quantity of MPL, M/C/AN was hydrated in triple-distilled water and digested with 10 N H2SO4 and 30% H2O2. After digestion, the samples were exposed to 0.2% ammonium molybdate and 15% ascorbic acid, and vortexed for 10 minutes. The absorbance was measured at 830 nm, and the concentration of MPL was calculated based on the standard curve of phosphorus and the chemical structure of MPL.18

Cellular uptake study

The cellular uptake of Ce6 was assessed by confocal microscopy and flow cytometry. B16F10 cells (a murine melanoma cell line; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Welgene, Daegu, Republic of Korea) supplemented with 10% of fetal bovine serum (FBS; GenDEPOT, Katy, TX, USA), and penicillin (100 unit per mL)/streptomycin (100 μg mL−1) (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). To visualize Ce6 uptake using a confocal microscope, B16F10 cells (5 × 104 cells per well) were seeded onto an 8-well cell culture slide (SPL Life Sciences, Pocheon, Republic of Korea). After 24 hours, the cells were incubated with nanoparticles at an AP concentration of 1 mg mL−1. After 3 hours, the cells were washed with PBS and fixed with 10% formalin in PBS for 10 min. The cells were then stained with Hoechst (2 μg mL−1 in PBS) and mounted with Fluoromount-G® (SouthernBiotech, Birmingham, AL, USA). Intracellular Ce6 was observed under a confocal microscope (Leica TCS SP8X; Leica Microsystems, Wetzlar, Germany). The cellular uptake of Ce6 was also quantified by flow cytometry. B16F10 cells (5 × 104 cells per well) were seeded onto 24-well plates (SPL Life Sciences) and treated as described above. The fluorescence intensity of Ce6 taken up by the cells was measured using a BD FACSCalibur flow cytometer (BD Bioscience, San Jose, CA, USA).

Detection of intracellular ROS

The generation of intracellular ROS was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Thermo Fisher Scientific, Inc.).19 B16F10 cells were seeded onto a 24-well plate and medium-suspended nanoparticles were applied at an AP concentration of 1 mg mL−1. After 3 hours, the cells were washed with PBS and irradiated with a 660 nm light emitting diode (LED) (Mikwang Electronics, Busan, Republic of Korea) at an intensity of 8000 mCd for 30 min. The irradiated cells were treated with 10 μM H2DCFDA for 10 min, and the fluorescence intensity was analyzed using a BD FACSCalibur flow cytometer.

In vitro assessment of photodynamic effects

In vitro anticancer effects were assessed by measuring the cell viability of nanoparticle-treated and -irradiated cells. B16F10 cells were seeded onto a 96-well plate, treated with medium-suspended nanoparticles at an AP concentration of 1 mg mL−1 for 3 hours, and irradiated with 660 nm of LED. One day later, cell viability was assessed by live/dead cell staining (live/dead viability assay kit; Thermo Fisher Scientific, Inc.) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) assays. Live cells and dead cells were stained with calcein acetoxymethyl and propidium iodide, respectively, and visualized by fluorescence microscopy (Leica DM IL LED; Leica Microsystems). Cell viability was also analyzed by MTT assay. Briefly, the cells were incubated with 250 μg mL−1 of MTT (10% of the total volume, dissolved in cell culture medium) for 2 hours, the supernatants were removed, and the formazan was dissolved in DMSO. Absorbance was measured at 570 nm.

Measurement of calreticulin exposure on cancer cells

Calreticulin exposure on PDT-exposed cancer cells was measured by flow cytometry.20 B16F10 cells were seeded onto a 24-well plate and treated with medium-suspended nanoparticles at an AP concentration of 1 mg mL−1. After 3 hours, cells were washed with PBS, irradiated with a 660 nm LED for 30 min, and further incubated for 8 h. The irradiated cells were collected and stained with an anti-calreticulin primary antibody (Santa Cruz Biotechnology) and then with Alexa Fluor 647-conjugated goat anti mouse IgG as the secondary antibody (BioLegend, San Diego, CA, USA). The expression level of calreticulin was measured using a BD FACSCalibur flow cytometer.

Animals and protocol approval

In this study, 5-week-old female C57BL/6 mice (Raonbio Inc., Yongin, Republic of Korea) were used. All animal experiments followed the Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources. The study protocol was approved by the Institutional Review Board for the use of animals at the College of Pharmacy, Seoul National University (approved experimental protocol number, SNU-180914-4).

In vitro assay of DC maturation and cytokine release

The in vitro adjuvant effects of nanoparticles were assessed by examining markers of mature DCs and the levels of cytokines released from bone marrow-derived DCs (BMDCs). BMDCs were obtained from a 5-week-old female C57BL/6 mouse (Raonbio Inc., Yongin, Republic of Korea). To prepare BMDCs, femurs and tibiae were isolated from the mouse, and the bone marrow was flushed out with an FBS-containing medium. Thereafter, the red blood cells were removed with lysis buffer (0.16 M NH4Cl, 14.2 mM NaHCO3, and 100 μM pH 8 EDTA) and the remaining monocytes were incubated for 7 days in a BMDC-specific medium composed of Iscove's modified Dulbecco's medium (IMDM, Welgene) containing 10% FBS, penicillin (100 units per mL)/streptomycin (100 μg mL−1), 20 ng mL−1 of recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF), 20 ng mL−1 of recombinant mouse interleukin-4 (IL-4; GenScript Biotech Corp., Piscataway, NJ, USA) and 50 μM β-mercaptoethanol (Sigma-Aldrich). Seven days later, immature DCs were seeded onto a 24-well plate at a density of 1 × 105 cells per well in DMEM containing serum and antibiotics. After overnight stabilization, the cells were incubated with nanoparticles at an AP concentration of 1 mg mL−1 for 48 hours. To analyze the maturation of BMDCs, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD11c antibody, allophycocyanin (APC)-conjugated anti-CD40 antibody, and APC-conjugated anti-CD86 antibody (BioLegend). The expression of BMDC maturation markers was assessed by flow cytometry. To assess the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) released from the nanoparticle-treated BMDCs, cell culture supernatants were examined using an ELISA kit (R&D Systems, Minneapolis, MN, USA).

Biodistribution of nanoparticles

The biodistribution of various nanoparticles was evaluated using a B16F10 tumor-bearing mouse model. To prepare the animal model, 5-week old C57BL/6 mice were subcutaneously injected with 2 × 105 B16F10 cells. Seven days after tumor inoculation, free Ce6, C/AN, and M/C/AN were intravenously injected at a dose of 10 μg of Ce6 per mouse. The biodistribution of Ce6 fluorescence was evaluated using IVIS® spectrum CT (PerkinElmer, Waltham, MA, USA).

In vivo assessment of DC maturation

The adjuvant effects of the nanoparticles were evaluated in the animal models via analysis of mature DCs in the spleen. Subcutaneous injection was used to generate B16F10 tumor-bearing C57BL/6 model mice (2 × 105 B16F10 cells per mouse). At 7 days post tumor inoculation, various nanoparticles were administered intravenously at an AN dose of 2.5 mg per mouse. One day after administration, tumor sites were irradiated at 660 nm for 30 min. Two days later, spleens were isolated from the mice and mature DCs were analyzed by flow cytometry-based assessment of CD40 and CD86 expression levels.

In vivo study of anticancer efficacy against primary and distant tumors

The in vivo anticancer effects of the nanoparticles were evaluated in 5-week-old C75BL/6 mice. The mice were subcutaneously injected in the right flank with 5 × 105 B16F10 cells to trigger the primary tumor formation. Seven days after tumor inoculation, nanoparticles were administered intravenously at an AN dose of 2.5 mg per mouse. One day after administration, tumors were irradiated with 660 nm LED light for 30 min. The distant tumor challenge was conducted in primary tumor-bearing mice on day 14 after primary tumor inoculation. B16F10 cells were inoculated subcutaneously into the left flank (1 × 105 per mouse). Tumor volumes were measured using calipers and calculated as: (major axis) × (minor axis)2 × 0.5.21

Tumor infiltrating lymphocyte assay

The population of cytotoxic T cells (CD3+CD8+) and regulatory T cells (Treg; CD3+CD4+CD25+FoxP3+) in the tumor was investigated in a B16F10 tumor-bearing C57BL/6 mouse model. The mice were subcutaneously injected in the right flank with 5 × 105 B16F10 cells. Seven days after tumor inoculation, nanoparticles were administered intravenously at an AN dose of 2.5 mg per mouse. One day after administration, tumors were irradiated with a 660 nm LED for 30 min. At 48 hours post-irradiation, tumors were extracted and stained with the FITC-conjugated anti-CD3 antibody, phycoerythrin (PE)-conjugated anti-CD4 antibody, peridinin–chlorophyll protein complex (PerCP)/cyanine (Cy) 5.5-conjugated anti-CD8a antibody, and PE/Cy5-conjugated anti-CD25 antibody (Biolegend). For intracellular staining of FoxP3 using an APC-conjugated anti-FoxP3 antibody (Thermo Fisher Scientific, Inc.), the tumor cells were fixed and permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Inc.). The stained cells were analyzed by flow cytometry.

In vivo toxicity study

The toxicity of the nanoparticles was assessed by histological study of organs. Five-week-old C75BL/6 mice were intravenously administered with nanoparticles corresponding to 2.5 mg of AN per mouse. After 24 hours, mice were sacrificed and perfused with 10 mL of PBS by intracardiac injection. Heart, lung, liver, kidney, and spleen samples were collected for hematoxylin and eosin staining.


Statistically significant differences among the groups were evaluated by a two-sided analysis of variance (ANOVA) with the Student–Newman–Keuls test. Calculations were performed using SigmaStat software (Systat Software, San Jose, CA, USA). A p-value less than 0.05 was considered statistically significant.

Results and discussion

Characterization of nanoparticles

The physicochemical properties of various nanoparticles were characterized by assessing the size, zeta potential, and morphology. The sizes of nanoparticles were not significantly affected by the entrapment of Ce6 and MPL. The mean sizes of plain AN, C/AN, and M/C/AN were 149.9 ± 12.9 nm, 150.0 ± 23.2 nm, and 164.2 ± 19.9 nm, respectively (Fig. 2A). Similarly, the zeta potentials of the nanoparticles were not significantly affected by the loading of Ce6 and MPL (Fig. 2B). TEM imaging revealed that plain AN and M/C/AN had similar morphologies (Fig. 2C). The loading contents of Ce6 per AN did not significantly differ between C/AN and M/C/AN (Fig. 2D), indicating that the co-loading of MPL to AN did not affect the loading amount of Ce6. The loading amount of MPL into M/C/AN was 12.8 ± 1.1 μg per mg of AN.
image file: c9bm01704f-f2.tif
Fig. 2 Characterization of M/C/AN nanoparticles. The physicochemical properties of the nanoparticles were observed in terms of the particle size (A) and zeta potential (B), and morphology (C). Scale bar, 200 nm. Quantification of Ce6 content in AP nanoparticles (D) (n.s., not significant).

AN was generated by the self-assembly of AP. This was enabled by the amphiphilic nature of AP, which has hydrophilic glutamic residues and hydrophobic phenylalanine residues. In aqueous solution, the hydrophilic glutamic residues are exposed to the solvent, while the hydrophobic phenylalanine forms the core. We previously reported that AN can be used to deliver an anticancer drug.21 In this prior study, paclitaxel was encapsulated at the hydrophobic core, while the nanoparticle surface was coated with photoresponsive polydopamine to enable photothermal chemotherapy.

Here, we observed that AN can be loaded with both Ce6 and MPL. Due to the hydrophobicity of Ce6 and MPL, they were expected to be loaded in the hydrophobic phenylalanine core of AN; this location appeared to provide sufficient room for co-entrapment, as the loading efficiency of Ce6 was not altered by the co-entrapment of MPL. In this study, we used Ce6 as a photosensitizer and co-entrapped it with the TLR4 agonist, MPL. However, AN can be used for the co-entrapment of other hydrophobic photosensitizers and immune adjuvants. Most photosensitizers suffer from low solubility in aqueous solution, and the entrapment of hydrophobic photosensitizers would be a promising strategy to overcome this solubility issue.

Uptake of Ce6 by B16F10 cells

The co-loading of MPL did not significantly affect the uptake of Ce6 in AN. The cellular uptake of Ce6 was investigated in nanoparticle-treated B16F10 melanoma cells. B16F10 cells treated with C/AN and M/C/AN showed similar intensities of Ce6-derived cellular fluorescence (Fig. 3A). Consistent with this observation, flow cytometry analysis revealed that the cell populations positive for Ce6-derived fluorescence were similar between the C/AN- and M/C/AN-treated groups (Fig. 3B).
image file: c9bm01704f-f3.tif
Fig. 3 Cellular uptake of Ce6 in M/C/AN nanoparticle-treated B16F10 cells. Cells were treated with nanoparticles, and intracellular delivery of Ce6 was observed using fluorescence microscopy (A). Scale bar, 50 μm. Fluorescence-positive populations were quantified by flow cytometry (B) (n.s., not significant; ***p < 0.001).

The mechanisms responsible for this high uptake of C/AN- or M/C/AN-delivered Ce6 will need to be studied in more detail. Previous reports showed that PGA-based nanoparticles are taken up by cancer cells via gamma-glutamyltransferase mediated endocytosis.22,23 Gamma-glutamyltransferase is known to be overexpressed in several cancer cell types, and to be involved in the progression of cancers.24,25 Since the outer layer of AN is composed of hydrophilic PGA, we speculate that C/AN and M/C/AN may be taken up via gamma-glutamyltransferase-mediated endocytosis.

Effect of 660 nm light irradiation on ROS levels and calreticulin exposure

We tested the effect of 660 nm light irradiation on ROS generation and calreticulin exposure in cells treated with the various nanoparticles. Unlike AN-treated cells, both C/AN and M/C/AN-treated cells showed substantial generation of intracellular ROS upon light irradiation (Fig. 4A). The presence of MPL in M/C/AN did not affect the generation of ROS upon light irradiation. The 660 nm irradiation also increased the exposure of calreticulin on the cell membrane: plain AN-treated cells did not exhibit calreticulin exposure, whereas C/AN- or M/C/AN-treated cells showed enhanced exposure of calreticulin after irradiation (Fig. 4B). The light-induced ROS-mediated anticancer effects were observed in the cells treated with C/AN and M/C/AN, but not in the cells treated with AN.
image file: c9bm01704f-f4.tif
Fig. 4 PDT-induced immunogenic cell death of M/C/AN nanoparticle-treated cells. Intracellular ROS production was assessed in B16F10 cells that were treated with nanoparticles and exposed to 660 nm light (A). Calreticulin expression was observed by flow cytometry analysis of cells that were treated with nanoparticles and exposed to 660 nm light (B). Live and dead cell populations were visualized among nanoparticle- and irradiation-treated cells (C). Scale bar, 50 μm. Viability of nanoparticle- and irradiation-treated cells by the MTT assay (D) (n.s., not significant; ***p < 0.001).

The fluorescence-based live/dead staining analysis revealed that there were higher populations of dead cells in the groups treated with C/AN or M/C/AN (Fig. 4C). Consistent with the fluorescence staining data, the MTT assay showed that cell viability was reduced to lower than 10% in cells treated with C/AN or M/C/AN and exposed to LED irradiation (Fig. 4D).

The enhanced exposure of calreticulin is likely to be related to the observed generation of ROS. Calreticulin is exposed on the cell surface under various stress conditions, including heat and ROS.21 The exposure of calreticulin in ROS-stressed cells has been reported by several groups.26,27 Calreticulin also acts as an “eat me” signal, as its exposure can promote the uptake of stressed cancer cells by immune cells (e.g., DCs) in the tumor microenvironment.28,29

In vitro DC maturation effect

Although C/AN and M/C/AN showed similar anticancer effects upon light irradiation, the two groups exhibited different DC maturation effects, as assessed using the DC maturation markers, CD40 and CD86. BMDCs treated with AN or C/AN did not exhibit induction of CD40 (Fig. 5A) or CD86 (Fig. 5B), whereas the M/C/AN-treated group showed enhanced expression of these DC maturation markers (Fig. 5A and B). The immune adjuvant activity of M/C/AN was further tested by assessing the cytokine-release patterns. Whereas BMDCs treated with AN or C/AN did not exhibit significant release of TNF-α (Fig. 5C) or IL-6 (Fig. 5D), those treated with M/C/AN showed 13.6-fold and 11.3-fold higher releases of TNF-α (Fig. 5C) and IL-6 (Fig. 5D), respectively, compared to the C/AN group.
image file: c9bm01704f-f5.tif
Fig. 5 Immune adjuvant effects of M/C/AN nanoparticles. Nanoparticle-treated bone marrow-derived DCs (BMDCs) were analyzed for populations positive for the DC maturation markers, CD40 (A) and CD86 (B). Cytokine secretion from BMDCs was measured by ELISA of TNF-α (C) and IL-6 (D). (n.s., not significant; ***p < 0.001).

The higher maturation of M/C/AN-treated BMDCs is likely to reflect the actions of MPL, which has been approved as an adjuvant for melanoma treatment. MPL is known to promote the maturation of DCs30via activating NF-κB31 and enhance the release of TNF-α and IL-6 from mature DCs.32

We observed that C/AN did not result in the activation of DCs (Fig. 5), even though it exposed calreticulin on cancer cells upon light irradiation (Fig. 4B). This observation is consistent with the results of previous studies showing that DAMP exposure alone could not induce a sufficient immune response, and that an immune adjuvant was needed to facilitate the activation of DCs.33,34 In a recent study, we found that the photothermal stimulus-triggered exposure of calreticulin on tumor cells increased their uptake by DCs, but had only limited effects on DC maturation.21 These studies suggest that the tumor cell uptake of DCs and the delivery of adjuvants are both important in the design of photoimmunotherapy.

Biodistribution and tumor accumulation

Molecular imaging revealed that both C/AN and M/C/AN accumulated in the tumor tissues. Since the fluorescence of Ce6 was used for molecular imaging, mice treated with free Ce6 were used for comparison. Compared to the free Ce6-treated group, the groups treated with nanoparticle-encapsulated Ce6 showed higher distribution in tumor tissues on whole-body imaging (Fig. 6A). Our ex vivo tumor tissue imaging also showed higher accumulation of Ce6 after administration in C/AN or M/C/AN compared to free Ce6 (Fig. 6B). Our quantitative image analysis showed that the C/AN group showed a 2.8-fold higher fluorescence intensity at the tumor tissues compared to the free Ce6 group (Fig. 6C). There was no significant difference in the tumor accumulation seen in the C/AN and M/C/AN groups.
image file: c9bm01704f-f6.tif
Fig. 6 Tumor accumulation of M/C/AN nanoparticles. One day after B16F10 tumor-bearing mice were injected with free Ce6 or various nanoparticles, the distribution of Ce6 was assessed by whole-body imaging (A). Tumors were obtained for imaging (B) and quantification (C) of Ce6 fluorescence. Results are expressed as mean ± SD (n = 3; n.s., not significant; ***p < 0.001).

The higher accumulation of C/AN and M/C/AN at tumor tissues is likely to at least partly reflect the enhanced permeability and retention effect.35,36 B16F10 tumors have also been reported to overexpress gamma-glutamyltransferase,37 suggesting that the gamma-glutamyltransferase-mediated endocytosis of C/AN or M/C/AN may have contributed to their tumor accumulation.24,25

The apparent lack of difference in the tumor accumulations of C/AN and M/C/AN may reflect that their outer surfaces are similar. Since the externally exposed glutamic acid residues of AN would be involved in gamma-glutamyltransferase-mediated endocytosis, both C/AN and M/C/AN may be taken up via a similar pathway.

In vivo activation of DCs by M/C/AN

Although C/AN and M/C/AN showed similar distributions to the tumor tissues, only M/C/AN activated DCs in vivo. Fig. 7A outlines the experimental schedule used to test the nanoparticle-mediated activation of DCs in vivo. In the spleen, the nanoparticle-induced activation of DCs was measured by assessing the CD11c+/CD40+ (Fig. 7B) and CD11c+/CD86+ (Fig. 7C) populations. Among the groups treated with the various nanoparticles, the M/C/AN group showed significantly higher expression levels of CD40 (Fig. 7D) and CD86 (Fig. 7E).
image file: c9bm01704f-f7.tif
Fig. 7 In vivo DC maturation in B16F10 tumor-bearing mice. The in vivo experimental schedule is illustrated in (A). Two days after 660 nm irradiation of tumor sites, CD40 (B and D) or CD86 (C and E) -positive DC populations were analyzed (B and C) and quantified (D and E) from untreated or nanoparticle-treated B16F10 tumor-bearing mice (data shown as mean ± SD, n = 4; *p < 0.05; ***p < 0.001).

The activation of DC by M/C/AN could be due to the immune-stimulating activity of MPL, which has been reported to stimulate DCs in vivo.30 Due to the hydrophobic nature of MPL, it has been formulated in liposomes,38 hydrogels,39 and polymeric nanoparticles.40 Our observation that M/C/AN activates DCs in vivo agrees with our finding that these particles have DC maturation effects in vitro (Fig. 5).

Antitumor effects against primary and rechallenged tumors

C/AN or M/C/AN treatment followed by 660 nm irradiation exerted different anticancer effects against primary and rechallenged B16F10 tumors. The experimental scheme for primary tumor inoculation, light irradiation, and distant tumor inoculation is shown in Fig. 8A. Both C/AN and M/C/AN effectively inhibited the growth of primary tumors upon 660 nm irradiation (Fig. 8B), whereas protection against tumor rechallenge was observed in the M/C/AN group but not the C/AN group (Fig. 8C). At day 40 after primary tumor inoculation, 100% survival was observed in the M/C/AN group, which exhibited complete protection against distant tumor formation. In contrast, the C/AN group exhibited 0% survival at day 40, due to the growth of distant tumors. In addition, no survival was seen in the other control groups, reflecting their primary and/or distant tumor burdens (Fig. 8D).
image file: c9bm01704f-f8.tif
Fig. 8 In vivo antitumor efficacy against primary and rechallenged tumors. The in vivo efficacy study was conducted as depicted in the schematic illustration (A). After treatment, the volumes of primary (B) and distant (C) tumors were monitored at an interval of 2–3 days (n.s., not significant; ***p < 0.001). Survival of mice was tracked for 40 days (D). The results are expressed as mean ± SD. n = 8 (untreated) and n = 5 (AN, C/AN, M/C/AN).

Although three mice of the control group (n = 8) showed notable tumor growth (Fig. 8C), no control mouse survived to the end of the study. Due to the rapid growth of B16F10 tumor cells, the survival rate was 62.5% at day 20, and 0% at day 35 for the control group (Fig. 8D). The anticancer effects of C/AN and M/C/AN against primary tumors are likely to reflect that entrapped Ce6 generates ROS upon light irradiation. Since both C/AN and M/C/AN contained similar amounts of Ce6 (Fig. 2), they exerted similar anticancer effects against the primary tumors. In contrast to the main killing mechanism of the primary tumors, which was red light-mediated PDT, the anticancer effect against rechallenged tumors was conferred by activation of immune systems.

Infiltration of CD8+ T cells to the tumor microenvironment

The treatment of mice with M/C/AN affected both cytotoxic T cell and Treg populations in the tumor microenvironment. The C/AN and AN groups exhibited similar CD3 and CD8+ T cell populations. Compared with the C/AN group, mice treated with M/C/AN showed significantly higher CD3 and CD8+ T cell populations in tumor tissues (Fig. 9A). Moreover, the groups treated with C/AN or AN showed similar CD3+, CD25+, CD4+ and FoxP3+ T cell populations compared to the untreated mice. However, the group treated with M/C/AN showed significantly lower populations of Tregs compared to the other groups (Fig. 9B). The CD8/CD4 ratio in the tumor microenvironment was 3.1-fold higher in the M/C/AN group compared to the untreated group (Fig. 9C).
image file: c9bm01704f-f9.tif
Fig. 9 Population of effector T cells in the tumor microenvironment. B16F10 tumor-bearing mice received nanoparticles intravenously. One day later, tumor sites were irradiated with a 660 nm LED. Two days after light irradiation, tumors were extracted and flow cytometry was used to analyze tumor-infiltrating lymphocytes, including CD3+CD8+ T cells (A), CD3+CD4+CD25+FoxP3+ T cells (B) and the CD8+/CD4+ T cell ratio (C). Representative flow cytometry data and the populations of T cells in the various treatment groups are presented (data shown as mean ± SD, n = 3; **p < 0.01; ***p < 0.001).

Our observations support the idea that the ability of M/C/AN to exert protective effects against tumor challenge reflects the PDT-induced immunogenic cell death of cancer cells, which is augmented by the MPL adjuvant. The mechanisms by which MPL increases CD8+ T cells and decreases Treg cells in the tumor microenvironment need to be studied further. However, we speculate that MPL promotes the maturation of DCs, which take up the ROS-induced calreticulin-exposed cancer cells and present the tumor antigens to the cytotoxic T cells. In the C/AN group, although calreticulin is exposed on the cancer cells, the lack of DC activation might limit the DC-mediated uptake and processing of tumor cells.

The increased exposure of calreticulin on tumor cells has been reported to promote the maturation of DCs to present cancer antigens to T cells.41 However, although tumor-associated antigens are taken up by DCs, they have relatively low immunogenicity and cannot induce potent immune responses.42 The activation of DCs by immune adjuvants, such as MPL, could enable DCs to induce potent immune responses. In our previous study, we reported that the delivery of imiquimod to DCs could enhance the presentation of tumor antigens to CD8+ T cells.21 In this study, the presence of MPL in M/C/AN could have stimulated the tumor antigen presentation of DCs.

Safety of M/C/AN

The in vivo safety of intravenously administered M/C/AN was tested by assessment of hematological parameters and histological staining. Hematological parameters, including white blood cell (WBC) counts, red blood cell (RBC) counts, and platelet (PLT) assessment, revealed no significant difference between the M/C/AN and untreated control groups (Fig. 10A).
image file: c9bm01704f-f10.tif
Fig. 10 Hematological parameters and histological staining. One day after injection of C/AN or M/C/AN nanoparticles, their toxicity was investigated by assessment of hematological parameters (A) and H&E staining (B). As a control, untreated mice were examined (data shown as mean ± SD, n = 3).

Histological staining showed that the intravenous treatment with M/C/AN did not cause pathophysiological signs in any major organs (heart, liver, spleen, kidneys, and lungs) (Fig. 10B).

The lack of notable acute toxicity conferred by M/C/AN is likely to reflect the biocompatible nature of AN, which is composed of PGA and phenylalanine, and is likely to be degraded to the natural amino acids, glutamic acid and phenylalanine. Previous studies reported that plain AP shows no notable evidence of hematological and organ toxicity.15,16 Previously, toxic effects were reported for both Ce6[thin space (1/6-em)]43 and MPL.44 In this study, however, these agents were distributed in the tumor tissues, which minimized their toxicity in various organs.


We herein demonstrate that a system for the co-delivery of a photosensitizer and an immune adjuvant could achieve anticancer effects against primary and rechallenged tumors. AP represents a naturally derived biopolymer that self-assembles into nanoparticles in aqueous solution and can entrap hydrophobic drugs in the nanoparticle core during this process. The entrapment of Ce6 in AN enabled the nanoparticles to exert a photodynamic anticancer effect via ROS generation upon 660 nm LED irradiation. The co-entrapment of MPL allowed the nanoparticles to modulate the immunological tumor microenvironment, and such particles protect the mice from tumor rechallenge. The entrapment of both photosensitizers and immune adjuvants in AN may have strong potential to be further developed for photodynamic immunotherapy.

Conflicts of interest

There are no conflicts to declare.


This research was supported by grants from the Ministry of Science and ICT, Republic of Korea (NRF-2018R1A2A1A05019203; NRF-2018R1A5A2024425; NRF-2018K2A9A2A 06019172, FY2018), and the Korean Health Technology R&D Project (No. HI15C2842 and HI19C0664), Ministry of Health & Welfare, Republic of Korea.


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These authors contributed equally to this work.

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