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https://doi.org/10.1038/s42003-023-05598-0

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Polphylipoprotein-induced autophagy mechanismwith high performance in photodynamic therapy

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Atsushi Taninaka1,2, Hiromi Kurokawa3, Mayuka Kamiyanagi1, Takahiro Ochiai2, Yusuke Arashida1,
Osamu Takeuchi1, Hirofumi Matsui3 & Hidemi Shigekawa 1 ✉Polphylipoprotein (PLP) is a recently developed nanoparticle with high biocompatibility andtumor selectivity, and which has demonstrated unprecedentedly high performance photosensitizer in photodynamic therapy (PDT) and photodynamic diagnosis. On the basis of these
discoveries, PLP is anticipated to have a very high potential for PDT. However, themechanism by which PLP kills cancer cells effectively has not been sufﬁciently clariﬁed. To
comprehensively understand the PLP-induced PDT processes, we conduct multifaceted
experiments using both normal cells and cancer cells originating from the same sources,
namely, RGM1, a rat gastric epithelial cell line, and RGK1, a rat gastric mucosa-derived cancerlike mutant. We reveal that PLP enables highly effective cancer treatment through PDT by
employing a unique mechanism that utilizes the process of autophagy. The dynamics of PLPaccumulated phagosomes immediately after light irradiation are found to be completely
different between normal cells and cancer cells, and it becomes clear that this difference
results in the manifestation of the characteristic effect of PDT when using PLP. Since PLP is
originally developed as a drug delivery agent, this study also suggests the potential for
intracellular drug delivery processes through PLP-induced autophagy.

1 Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan. 2 TAKANO Co. LTD. Miyada-mura, Kamiina-gun,
Nagano 399-4301, Japan. 3 Fuculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. ✉email: hidemi@ims.tsukuba.ac.jp

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hotodynamic therapy (PDT) treats diseases by dosing
photosensitizers into a body and irradiating them with lightto generate reactive oxygen species (ROS)1–3. It is noninvasive with few adverse effects and exhibits a high therapeutic
effect on lesions that are difﬁcult to treat by surgical techniques. It
is used not only for cancer therapy but also for conditions such as
age-related macular degeneration and skin diseases4–6. Recently,
antimicrobial PDT has been developed as a method to efﬁciently
inactivate pathogens without inducing resistant bacteria, and it
has been applied to treat periodontal disease7–10. It also has beenreported that PDT has an abscopal effect: the shrinkage ofuntreated tumors was concurrently observed with the shrinkageof tumors upon local treatment11–13.
The observed effect of PDT mainly depends on the chemical
properties of the photosensitizer. Therefore, various photosensitizers suitable for treatment with PDT, for example, porﬁmer
sodium and talaporﬁn sodium, have been developed14. Polphylipoprotein (PLP) is a recently developed nanoparticle with highbiocompatibility and tumor selectivity11,15,16, which has demonstrated unprecedentedly high performance as a photosensitizer in
PDT and photodynamic diagnosis (PDD). Figure 1a shows the
structure of PLP. It is a porphyrin-base nanoparticle consisting ofimitated lipoprotein formed by lipids and proteins, with a coreshell structure, hydrophilic functional groups, and hydrophilicmolecules on the outside hydrophobic functional groups andhydrophobic molecules on the inside. Pyropheophorbide-lipid, acomponent of PLP, has phospholipids and a chlorin ring andforms a spherical structure with the hydrophobic chlorin ringarranged inside. Although PLP was originally developed as a drugdelivery agent15,16, it has been investigated as a photosensitizer
with high performance in PDT and PDD.
PDT using PLP, similarly to conventional PDT, generates ROS
by dosing cells with PLP followed by light irradiation, which isconsidered to destroy lesions and exert a therapeutic effect(Fig. 1b). However, this mechanism does not provide sufﬁcientunderstanding of why PLP enables the highly effective treatmentof cancer cells in PDT. Recently, it has been revealed that PLPs
accumulate on the phagosome membrane in the cells of RGK1, a
rat gastric mucosa-derived cancer-like mutant17.
Phagosomes are vesicles in which parts of cytoplasm and foreign substances are surrounded by a phospholipid bilayer membrane called an isolation membrane and act in the process of
autophagy18,19. Autophagy, the function of cells to degrade theirunnecessary components, has a mechanism that begins with thegeneration of phagosomes. When PLPs are taken into a cell, they
may be considered as targets to be degraded and sequestered byphagosomes17. If phagosomes accumulate PLPs, according to the
autophagy mechanism, the PLP-accumulated phagosomes maybind to lysosomes, forming phagolysosomes, which normallydegrade the taken substances using hydrolytic enzymes in thephagolysosomes. Figure 1c shows an overlap image of bright-ﬁeld
and ﬂuorescence images of a PLP-dosed RGK1 sample. Figure 1dshows a series of z-stack ﬂuorescence images of the phagosomes
shown in Fig. 1c obtained by a confocal super-resolution
microscope (left) in this study and a three-dimensional (3D)
image produced from the z-stack images (right). As expected, thephagosome-lysosome combined structure is clearly visible.
If PDT is performed after phagolysosomes with accumulated
PLPs are produced in a cell, the ROS generated by light irradiation destroys the membrane of the phagosomes, then, foreign
substances the phagolysosomes, such as hydrolytic enzymes and
ROS, diffuse into the cell, destroying organelles17. By thesemechanisms, the cell undergoes necrosis, as shown in Fig. 1b17,20.
This process convincingly explains why PLP demonstrates higher
efﬁcacy than other photosensitizers in PDT. However, sincephagosomes are produced in all types of cells, such as cancer cells

Fig. 1 Schematic diagram of PDT with PLP. a Structure of polphylipoprotein (PLP). b Phase-contrast images of RGK1 immediately after and 30 min after
irradiation with light of 655 nm for 1 min. After 30 min, blebs were formed, and the cells became necrotic. c Overlap image of bright-ﬁeld and ﬂuorescence
images of a PLP-administered RGK1 sample. The ﬂuorescent images are pseudocolored in red. d Series of z-stack ﬂuorescence images of a phagosome
after PLP administration (left) and three-dimensional (3D) image formed using the z-stack images (right). The structure of phagolysosome, the binding
structure between phagosome and lysosome, is clearly visible, where the lysosome is indicated by the white arrow.
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Fig. 2 Phase-contrast images. a RGK1 and b RGM1 after irradiation of 28.7 mW cm−2 light with a central wavelength of 655 nm for 1 min. c, d Enlarged
images of the squared areas in a and b, respectively. As evident in the enlarged images, from 0 to 5 min, the cells maintain the same shape. However, after
that, in RGK1, the shape becomes rounded, indicating that RGK1 has sustained damage.

and normal cells, this mechanism of necrosis alone cannot
explain the selective tumor treatment by PDT with PLP, which ishighly effective only for cancer cells. Since PLP is a promising
photosensitizer, clariﬁcation of its effective role in PDT is strongly
desired.
To respond to this urgent requirement, we conducted multifaceted experiments using both normal cells and cancer cells
originating from the same sources, namely, RGM121, a rat gastricepithelial cell line, and RGK121, a rat gastric mucosa-derived
cancer-like mutant. The experiment comparing the effects of PDT
using normal cells and cancer cells is a ﬁrst-of-its-kind attempt.
We revealed that PLP has a previously unreported mechanismthat enhances the effectiveness of cancer treatment by inducing
and utilizing autophagy. PLP-induced phagosome generation andits reaction processes upon light irradiation in PDT show entirely
different behaviors in RGK1 and RGM1, explaining themechanism of PDT by PLP with highly selective efﬁcacy on
cancer cells. Since PLP was originally developed as a drug delivery
agent, this study also suggests the potential for intracellular drugdelivery processes through PLP-induced autophagy.
Results
Sample preparation. RGM1 and RGK1 samples were prepared inΦ = 60 mm culture dishes with 30–50% cell area occupancy twodays after passage. Details of the culture method are described in
Methods. PLP was added to samples to a ﬁnal concentration of
19 μM, and after allowing the samples to stand in an incubator for
24 h, they were washed with a new medium to remove the PLPs
not taken up into the cells. The samples were placed in a top-stage
incubator on an IX83 optical microscope system (Olympus
Corp.), and experiments were carried out. For PDT measurements, samples were irradiated with light with wavelengths from
635 to 675 nm through a bandpass ﬁlter.

Effect of PLP-PDT at the cell level. First, we observed theselective effects of PLP-induced PDT at the cell level. Figure 2
shows phase contrast images of (a) RGK1 and (b)RGM1 samples, which were irradiated with light of 28.7 mW cm2 for 1 min. As shown in Fig. 2a, there was no deformation of the
RGK1 at 5 min after the irradiation, but shrinkage was observed
at 15 min. Thereafter, the cells had become rounded and werelifted off the bottom of the dish at 60 min (Fig. 2c). In contrast, asshown in Fig. 2b, there was no deformation of the RGM1 even at
60 min after the irradiation, and all cells migrated as usual(Fig. 2d). These results indicate that although the RGK1 cells
were damaged, the RGM1 cells were undamaged by the PDT
process.
Since singlet oxygens (1O2), which are generated fromphotosensitizers by the PDT process, are transformed to othertypes of ROS and are considered to damage several cytoplasmic
organelles2,14,17,20,22,23, the amount of photosensitizer s introduced into the cells may inﬂuence the effect of PDT observed forcancer and normal cells17,20. In fact, PDT with talaporﬁn sodium
uses the difference in its amount taken up in cancer and normal
cells. Namely, the PDT treatment is performed when the
difference in the amounts of talaporﬁn sodium in cancer andnormal cells is at its maximum20. Therefore, the same amounts of
PLP were dosed into the RGK1 and RGM1 samples shown in
Fig. 2. Namely, the incubation time after dosing PLP was set to
24 h, longer than the 3 h at which the difference in the amounts
taken up is maximum. Figure 3 shows the ratios of intracellular
PLP dosed into RGK1 and RGM1, determined from theﬂuorescence intensity of PLP (See Methods for detail). They
were estimated to be 3.6% and 2.7%, respectively, and no
signiﬁcant difference in the observed PDT effect was obtained.
The difference in the effects of PDT with PLP on cancer cells
and normal cells shown in Fig. 2 was conﬁrmed at the cellularlevel. We next investigated the cause of this difference.

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PLP distributions in RGK1 and RGM1. One possible factor isthe difference in the location of PLP accumulated in cells. The
lifetime of 1O2 produced in cells by the PDT process is short; theyreadily oxidize surrounding substances or are transformed into
other ROS2,14,22,24. Therefore, the organelles where PLPs accumulate are rapidly affected. In particular, the generation of ROS
near the organelles, such as cell nuclei and mitochondria, causes
severe damage to biological activities. Therefore, to clarify thelocation of the dosed PLPs, we carried out ﬂuorescence measurements using a confocal super-resolution microscope. Figure 4
shows the results, where a and f are endoplasmic reticulum (ER)ﬂuorescence images, b and g are mitochondrial ﬂuorescence
images, c and h are PLP ﬂuorescence images, d and i are merged
mitochondrial and PLP images, and e and j are enlarged images ofthe squares in d and i for the RGK1 and RGM1 samples,
respectively.
In the case of RGK1, the PLP ﬂuorescence image contains brightspots and ring-shaped structures (inset of Fig. 4c)17, which arelocalized near mitochondria close to the nucleus, as shown in
Fig. 4d, e. Because previous studies have shown that phagosomes
are generated around the ER and mitochondria25, and that PLPs

Fig. 3 Ratio of PLP taken into cells to the dose for RGK1 and RGM1. Eachamount of PLP was determined by the ﬂuorescence intensity of PLP(N = 24). The rates are 3.6% for RGK1 and 2.7% for RGM1.

accumulate in phagosomes for RGK117, the bright spots and ringshaped structures observed here are considered to be phagosomes.
The ring shape correspond to the cross section of the sphericalstructure of phagosomes obtained by confocal super-resolution
microscope measurement, as shown in Fig. 1d18. In contrast, in thecase of RGM1, although similar bright spots and ring-shaped
structures, which are considered to be phagosomes, were formed, as
shown in the inset of Fig. 4h, most of them are spread out in a band
shape at the edge of the cell and do not overlap with the
distribution of mitochondria, as shown in Fig. 4i, j.
Since PLPs are expected to distribute uniformly within cells
when added, the observed difference in the distribution isconsidered to have occurred during the 24 h while the samples
were allowed to stand in the incubator. Namely, some mechanismof autophagy may have caused the difference in the PLPaccumulated phagosome distribution. Therefore, we conducted
experiments to further clarify the observed processes.
PLP-induced phagosome generation and PLP capture processes. First, to determine the formation processes of PLPaccumulated phagosomes, measurements with the autophagy
detection reagent DAPGreen, which emits ﬂuorescence whenincorporated in the autophagosome membrane, and PLP ﬂuorescence were performed. Figure 5 shows the results, where acomprises ﬂuorescence images of RGK1 administered with
DAPGreen ﬁrst and then PLP, b comprises ﬂuorescence imagesof RGK1 administered with PLP and then DAPGreen, c comprises ﬂuorescence images of RGM1 administered with DAPGreen and then PLP, and d comprises ﬂuorescence images of
RGM1 administered with PLP and then DAPGreen.
For RGK1, when DAPGreen was administered ﬁrst, thelocations of DAPGreen and PLP ﬂuorescence in the images were
almost the same, as shown in Fig. 5a. They were observed as ringsof various sizes with diameters of 0.3–2 μm, similarly to in Fig. 4.
For RGM1, as similarly shown in Fig. 5c, the locations of
DAPGreen and PLP ﬂuorescencewere almost matched. However,unlike the case of RGK1, they were observed as ring structureswith almost the same diameter of about 0.3 μm.
In contrast, when PLP was administered ﬁrst, as shown in
Fig. 5b, d, the locations of DAPGreen and PLP ﬂuorescence did

Fig. 4 Super resolution ﬂuorescence images. a, f Endoplasmic reticulum (ER) ﬂuorescence images, b, g mitochondrial ﬂuorescence images, c, h PLP
ﬂuorescence images, d, i merged ER, mitochondrial, and PLP images, and e, j enlarged images of the squares in d and i for the RGK1 and RGM1 samples,
respectively. Red ring-shaped structures are observed in the insets of c and h and in e and j. ER-Tracker™ Green (Invitrogen™) and MitoTracker™ Orange
CMTMRos (Invitrogen™) were used for ﬂuorescence observation of the ER and mitochondria, respectively.
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Fig. 5 Double staining experiment with the autophagy detection reagent DAPGreen and PLP. a Fluorescence images of RGK1 administered with
DAPGreen ﬁrst and then PLP, b ﬂuorescence images of RGK1 administered with PLP and then DAPGreen, c ﬂuorescence images of RGM1 administeredwith DAPGreen and then PLP, and d ﬂuorescence images of RGM1 administered with PLP and then DAPGreen. Yellow arrows indicate lysosomes.
DAPGreen is a ﬂuorescent probe that increases its ﬂuorescence in response to a lipid-soluble environment. When DAPGreen is incorporated into thephagosome membrane, the lipid environment may change, leading to an increase in ﬂuorescence. Indeed, in Fig. 5a, c, the phagosomes are stained with
DAPGreen. On the other hand, in Fig. 5b, d, since the phagosomes do not take up PLP, the luminescence of DAPGreen does not match that of thephagosomes induced by PLP, and it is dispersed in a cell. In the case of Fig. 5b, d, as observed in the magniﬁed images, a high-contrast, island-like structure
was observed in RGK1, suggesting the effects of differences in the intracellular lipid-soluble environment. Phagosomes use the endoplasmic reticulum
membrane as a material; therefore, they might be structurally related to the endoplasmic reticulum. Details exceed the scope of this paper, so we leave it asa topic for future research and will not delve further into it here.

not match. In addition, the ring-shaped structures were observedin the PLP ﬂuorescence measurement but not in the DAPGreen
ﬂuorescence measurement. These results indicate that thegeneration of phagosomes is triggered by PLP. In addition, inthe case of RGK1, similarly to in previous results17, ring-shaped
phagosomes are combined with lysosomes (indicated by yellow
arrows in the ﬁgure) to form phagolysosomes, as shown in Fig. 5.
On the other hand, for RGM1, only the ring-shaped phagosomes
were observed, and no combination with lysosomes occurred.
These results are summarized as follows:(1) PLP initiates phagosome generation.
(2) For both RGK1 and RGM1, PLP is incorporated inphagosome membranes.
(3) The phagosomes in the RGK1 sample are large (0.3–2 μm)
and located close to the cell nucleus and mitochondria,
whereas the phagosomes in the RGM1 sample are small(0.3 μm), uniform in size, and located at the edge of the celland spread out in a band shape.
(4) Phagolysosomes are formed in RGK1 but not in RGM1.
Regarding ﬁndings (1) and (2), the model shown in Fig. 6 isconsidered to explain the PLP-induced phagosome generation
and PLP uptake. As shown in Fig. 6a, if DAPGreen isadministered ﬁrst, it remains unchanged in the cells because it
does not induce phagosome generation. Then, when PLPs areadministered subsequently, phagosome generation is induced.
Phospholipids are supplied from the ER to phagosome
precursors25–28, and DAPGreen is incorporated in the phagosome membrane together with PLPs29. Therefore, phagosome
membranes are stained with DAPGreen and PLP after thephagosomes are formed. Namely, the locations of DAPGreen and
PLP ﬂuorescence are almost the same.
On the other hand, as shown in Fig. 6b, when PLP isadministered ﬁrst, phagosome generation is induced, andphospholipids are supplied from the ER to the phagosome
precursors. Phagosomes take up PLP; thus, the phagosome
membrane is stained with PLP after the phagosome are formed.
Therefore, even after subsequent DAPGreen administration,
the completely formed phagosomes do not incorporate DAPGreen. As a result, the phagosomes do not ﬂuoresce with
DAPGreen but with PLP, and, therefore, the locations of
DAPGreen and PLP ﬂuorescence do not match, as shown in
Fig. 5b, d. PLP was found to be the factor that induces
phagosome formation and to selectively accumulate in thephagosome membranes. As a result, the ring-shaped phagosome structure was observed in the ﬁne z-stack structure shownin Fig. 1d. Time-lapse images obtained by a confocal superresolution microscope using PLP ﬂuorescence were later used
to observe the light-induced dynamics of PLP-accumulated
phagosomes in PDT.

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Fig. 6 Model to explain the uptake of DAPGreen and PLP into the phagosome membrane. a DAPGreen administered ﬁrst and b PLP administered ﬁrst.

Size and distribution of PLP-accumulated phagosomes.
Regarding the size difference indicated in ﬁnding (3), RGK1 hasheterogeneous phagosome sizes, with many phagosomes
exceeding 1 μm in diameter, whereas RGM1 has more uniform
sizes with diameters around 0.3 μm. Although details have not yet
been clariﬁed, the phagosome size is determined by the expression level of ERdj8, a protein of the ER membrane30. Therefore,
the expression levels of ERdj8 in RGK1 and RGM1 were compared by immunoﬂuorescent staining.
Figure 7a, c shows phase-contrast images of RGK1 and RGM1,
and Fig. 7b, d shows immunoﬂuorescence staining images of
RGK1 and RGM1, respectively. Figure 7e represents the average
ﬂuorescence intensities of ten cells in Fig. 7b, d. The average
ﬂuorescence intensity of RGK1 is 11 % higher than that of RGM1.
From this comparison, it can be inferred that RGK1 has a higher
expression level of ERdj8 than RGM1, supporting the observed
difference in the phagosome size.
Regarding the distribution difference indicated in ﬁnding (3),as was pointed out in the previous section, since PLP is expected
to distribute uniformly within cells when added, the observed
difference in the distribution is considered to occur during the
24 h while the samples were allowed to stand in the incubator.
Namely, the PLP-accumulated phagosomes may have moved
toward the center of cells in RGK1 and toward the edge of thecells in RGM1.
In autophagy, the state of cellular starvation affects theprocess31–35. Motor proteins drive phagosomes to travel along
microtubules, the main intracellular transportation routes withina cell36–43. When a cell is in a state of starvation, a phagosome
merges with a lysosome to form a phagolysosome18,44–48. The
lysosome is a digestive organ of the cell, breaking down andreusing molecules such as parts of organelles, proteins, lipids, andsugars taken up by the phagosome.
Moreover, the phagosome membrane contains LC3 (microtubule-associated protein light chain 3)40,49,50, which binds tomicrotubules through the scaffolding protein JIP1 (JNK-interacting protein 1)40. JIP1 was originally identiﬁed for its ability torecruit multiple kinases in the JNK pathway, it had beensuggested
that
JIP1
regulates
constitutive
axonaltransport36,39–43,51. If the cell is a state of starvation, phagosome
tends to move toward the centrosome along the
microtubules31,32,34,35. If the cell is not in a state of starvation,
phagosome tends to move toward the edge of the cell along the
microtubules31,32,34–38,40,41,52. This mechanism is consistent withthe observation that phagosomes accumulate at the center in
RGK1 but localize at the edge of the cell in RGM1.
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Furthermore, it is known that STX17 (Syntaxin 17), a
membrane protein, plays a crucial role in the formation ofphagolysosomes33,48,53. In a state of starvation, STX17 reduces its
localization to mitochondria and ER and accumulates on thephagosome membrane;33,53 thus, the phagosome binds to thelysosome via STX1748. On the other hand, when the cell has
sufﬁcient energy, little STX17 accumulates on the phagosome
membrane, making it difﬁcult for the phagosome to bind to thelysosome. This mechanism aligns well with the observation ofphagolysosomes in RGK1, and the phagosomes remain in RGM1.
From the above considerations, if cancer cells are in a state of
starvation, the formation and distribution of phagosomes andphagolysosomes in RGK1 and RGM1 can be well explained. To
determine the starvation status of RGK1 and RGM1, we
examined the activity of AMPK (adenosine monophosphateactivated protein kinase) in the cells. It is known that when cells
are in a state of starvation, such as when there is insufﬁcient ATP,AMPK is phosphorylated (activated) to become pAMPK54.
Therefore, we performed western blot measurements of
pAMPK/AMPK in RGK1 and RGM1. Figure 8 comprises (a) arepresentative western blot image and (b) the ratio of pAMPK to
AMPK estimated from the western blot. As shown in Fig. 8b, theactivity of pAMPK is high in RGK1. That is, the result that
phagolysosomes are formed only in RGK1 aligns well with theinterpretation that the RGK1cancer cells are in a state of
starvation.
Light-driven dynamics of PLP-accumulated phagosomes in
PDT. Finally, to examine in detail the effect of PDT observed at
the cellular level shown in Fig. 2 at the level of the phagosome,
we observed the dynamics of phagosomes during 1 min lightirradiation. Figure 9a, b shows the time-lapse images obtainedfor RGK1 and RGM1, respectively. As shown in Fig. 9a, phagolysosomes, namely, phagosomes bound to lysosomes indicatedby yellow arrows, maintained their spherical shape while
deforming up to 50 s, after which they were destroyed. In contrast, as shown in Fig. 9b, small phagosomes fused with thesurrounding phagosomes during the ﬁrst 10 s bind to becomelarger ones with a diameter of 0.5–1 μm. They were preserved
and not destroyed.
Here, upon light irradiation, singlet oxygen was generated by
the chlorin ring of pyropheophorbide-lipid accumulated in thephagosome membrane, and the phagosome membrane wasoxidized and deformed. After 50 s, the cage structure of thephagolysosome could no longer be maintained and was

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Fig. 7 Analysis of the expression level of ERdj8, a protein of the ER membrane. a, c Phase contrast (PH) and b, d immunoﬂuorescent (IF) staining imagesof RGK1 and RGM1, respectively. e Average ﬂuorescence intensities of ten cells in b and d. The average ﬂuorescence intensity of RGK1 is 11 % higher thanthat of RGM1. From this comparison, it can be inferred that RGK1 has a higher expression level of ERdj8 than RGM1, supporting the observed difference inthe phagosome size.

Fig. 8 Western blot analysis of AMPK-activated protein kinase α (AMPK) and its phosphorylated (activated) form α (pAMPK) for RGK1 and RGM1.
a Representative western blot images. Western blot images including protein molecular weight markers are shown in Supplementary Fig. S1. b Ratio of
pAMPK to AMPK estimated from the western blot images. **p < 0.01.

destroyed. Thereby, the internal hydrolases and ROS leaked out,
damaging the cell17.
In contrast, in the case of RGM1 phagosomes shown in Fig. 9b,
the administered PLP induced the generation of small phagosomes as indicated by the low expression level of ERdj830.
Phagosome precursors surrounded the PLP and concomitantly
accumulated the PLP in the phagosome membrane. When PDT
was performed and light was irradiated, the surrounding

phagosomes fused and transformed into larger phagosomes
within the ﬁrst 10 s. Since the lysosomes did not bind tophagosomes, ROS and hydrolytic enzymes did not leak into thecell during fusion. After 10 s, the phagosomes maintained theirspherical shape and size and did not break; thus, they did not
damage the cells.
The size of the completed phagosome depends on theexpression level of ERdj8, it is dependent on the normal state

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Fig. 9 Time-lapse images of phagosomes with PDT. a RGK1 and b RGM1 phagosomes during 1 min light irradiation (337 mW cm-2). The frame rate is 2
fps. In RGK1, the phagolysosome, which was formed by the fusion of the phagosome indicated by the red arrow and the lysosome indicated by the yellow
arrow, maintained its spherical shape for up to 50 s, then was destroyed. In RGM1, small phagosomes fused with the surrounding phagosomes to becomelarger ones with a diameter of 0.5–1 μm during the ﬁrst 10 s. After 10 s, the phagosomes maintained their spherical shape and size and did not break; thus,
they did not damage the cells. Supplementary Movie S1 in the Supplementary shows this time variation.

in which the cells are not exposed to oxidative stress. Therefore, it
is suggested that the observed change in the size of phagosomes is
considered to be subjected to oxidative stress. As shown in
Fig. 9b, the phagosomes did not grow indeﬁnitely during the lightirradiation, and their growth stopped after about 20 s. This
cessation of growth may be due to physical or structurallimitations, such as phospholipid packing due to oxidation of
the phagosome membrane.
These factors are considered to be responsible for the observed
differences in the effects of PLP-PDT on RGK1 and RGM1.
Discussion
On the basis of the obtained results, the dynamics shown in
Fig. 10 is considered to explain the PDT effect when using PLP. In
both cases, administered PLP induces phagosome generation,
where PLP accumulates on the phagosome membrane. The
phagosome size depends on the level of ERdj8 expression, withlarger phagosomes (0.3 ~ 2 μm) formed in RGK1 and smaller
phagosomes (~0.3 μm) in RGM1.
As explained in the section titled “Size and distribution of
PLP-accumulated phagosomes” in the main text: (1) Phagosomes
induced by PLP are widely distributed within the cell upon
formation. Since RGK1 is a cancer cell and in the starved state,
they move towards the near the cell nucleus, while in RGM1 is anormal cell and not in the starved state, they move towards theperiphery. (2) Lysosomes are formed by the Golgi apparatus andhence are mainly found near the nucleus, though some may belocated at the periphery. (3) Phagosomes only bind with lysosomes once STX17 localizes on their membrane. (4) STX17 exists
at the mitochondria and the endoplasmic reticulum and islocalizes on phagosomes in the starved state (RGK1). (5)Therefore, it is likely that RGM1 is not in a starved state as
conﬁrmed in Fig. 8 and phagosomes rarely bind with lysosomes.
(6) Lysosomes only incorporate and emit light from PLP after
binding with phagosomes. Thus, the probability that the brightspots in RGM1 are lysosomes is low. Also, in RGM1, smaller8

phagosomes combine to grow in size. This result suggests
overlapping regions between adjacent phagosomes can be seen as
bright spots. Smaller phagosomes can be seen as bright spots,
too. Similarly, in RGK1, small phagosomes or overlapping
regions between adjacent phagosomes might also be visible asbright spots.
The brightness of the emission depends on the integrated valueof PLP-induced luminescence in the z-direction. The superresolution confocal microscope used in our experiments has ahigh resolution in the z-direction, which means only the focusedlayer is visible. Phagosomes appear ring-shaped due to theirspherical membrane structure, and their shape changes when the
focal point shifts due to factors like drift. While phagosomes andlysosomes merge by binding their membranes, the lysosome
portion appears as a small clump ﬁrst. This process may lead to alarger integrated value in the z-direction, making it seem brighter.
Smaller phagosomes similarly appear as bright spots.
When PDT is performed under such conditions, in RGK1, themembrane of the phagosome ruptures and its contents are
released. The released contents considerably damage the cell,
leading to the necrosis shown in Fig. 2. In contrast, in RGM1,
smaller phagosomes merge to form larger ones. During theexperiment, the phagosomes were not destroyed. For PDT using
PLP, a new mechanism utilizing the autophagy pathway was
discovered. This ﬁnding provides a new understanding of various
aspects of the treatment, including the highly selective andpotential effects of PLP-PDT.
Autophagy, which serves to normalize cellular functions byremoving misfolded proteins, among other substances, has atumor-suppressing effect in early-stage cancer55–58. Therefore,
inducing the formation of phagosomes can lead to tumor suppression. On the other hand, in growing tumors, autophagy acts
as a crucial survival pathway, providing nutrients and eliminating
anti-cancer agents55–57,59. Therefore, controlling the autophagy
in tumors can be an effective treatment55–59. Moreover, thecombination of PLP and PDT, which selectively controls

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Fig. 10 Schematic diagrams to explain the phagosome generation and reaction processes in PDT. a RGK1 and b RGM1. In both cases, administered PLPs
induce phagosome generation, where PLPs accumulate on the phagosomal membrane. The phagosome size depends on the level of ERdj8 expression, as
shown in Fig. 7, with larger phagosomes (0.3–2 μm) in RGK1 and smaller phagosomes (~0.3 μm) formed in RGM1. The phagosomes move along the
microtubules. In RGK1, under starvation conditions, they move towards the cell center, merging with lysosomes to form phagolysosomes, leading to thedigestion of their contents. In RGM1, they move toward the periphery of the cell and do not merge with lysosomes. When PDT is performed under suchconditions, in RGK1, the membrane of the phagosome ruptures and its contents are released. The released contents considerably damage the cell, leading
to necrosis, as shown in Fig. 2. In contrast, in RGM1, smaller phagosomes merge to form larger ones. During the experiment, the phagosomes were notdestroyed. These processes are considered to be responsible for the difference in PDT observed in Fig. 2.

phagosomes, can potentially contribute to the treatment of various diseases associated with autophagy.
In conclusion, we have revealed that PLP enables highly
effective cancer treatment through PDT by employing a previously unreported PDT mechanism that utilizes the process of
autophagy. We revealed that PLP enables highly effective cancer
treatment through PDT by employing a unique mechanism thatutilizes the process of autophagy. The dynamics of PLPaccumulated phagosomes immediately after light irradiation
were found to be completely different between normal cells andcancer cells, and it became clear that this difference results in the
manifestation of the characteristic effect of PDT when using PLP.
Since PLP exhibits luminescence of sufﬁcient intensity evenafter its accumulation on a phagosome membrane, PLP is
expected to play an important role in therapies targeting autophagy and as a highly efﬁcient photosensitizer for PDT. PLP was
originally developed as a drug delivery agent. This study revealed
the potential for intracellular drug delivery processes through
PLP-induced autophagy. This discovery could potentially pave
the way for entirely new possibilities in the future. Other biological materials might also possess this mechanism. However, toexhibit effects like PLP, it is considered that the following conditions must be met: (1) They must have a structure that allows
efﬁcient uptake by cells. (2) They must be substances that induce
autophagy. (3) They must be materials that accumulate on thephagosome membrane. Further research into these aspects isdesirable.
Methods
Preparation of polphylipoprotein (PLP). PLP is a photosensitizer that contains a hydrophobic drug-loadable core enveloped in a porphyrin–lipid monolayer and constrained by ApoA-1
mimetic
R4F(Ac-FAEKFKEAVKDYFAKFWD)
peptidenetworks16,17,20.
Culturing cells. RGM1, a rat gastric epithelial cell line, waspurchased from RIKEN CELLBANK (Ibaraki, Japan). The
RGK1 cells used in this study were rat gastric mucosa-derived

cancer-like mutant cells21, a chemically induced oncogenic
cancer-like mutant of RGM121. RGK1 cells were cultured in
DMEM/F12 without L-glutamine (Sigma-Aldrich Japan K.K.,
Tokyo, Japan). RGM1 cells were cultured in DMEM/F12 with
L-glutamine (GibcoTM, Thermo Fisher Scientiﬁc K.K.).
Sample preparation. PLP (ﬁnal concentration 19 μM) was added
to RGK1 and RGM1 cells cultured in a plastic dish of 60 mm
diameter (for phase-contrast observation) or a glass bottom dishof 35 mm diameter (for super-resolution observation) andallowed to stand for 24 h in an incubator at 37 °C with a CO2
concentration of 5%. This concentration of PLP is not toxic20. In
a previous experiment in which the incubation time was varied20,
it was conﬁrmed that PLP dose does not saturate in 3 h but
saturates in 12 h. In this study, we performed incubation for 24 hso that the amount of sensitizer entering the cells was saturate.
For talaporﬁn sodium, for example, the quantum yield is the same
in both normal and cancer cells, and the difference in uptake isbeing utilized14. In this study, we adjusted the amount of PLP
taken up by RGK1 and RGM1 to be equivalent. When PLP isincorporated into the phagosomal membrane, it is degraded. As
shown in Fig. 3–5, there is no signiﬁcant difference in luminescence intensity when using PLP in RGK1 and RGM1. This result
suggests that the quantum yield of PLP is almost equivalent. The
experiment’s validity, where the uptake amount of PLP was
adjusted for RGK1 and RGM1 to make the concentrations of both
equivalent, is supported.
Measurement of PLP concentration in a cell. RGK1 and RGM1
were cultured in a 24-well-plate, then PLP (ﬁnal concentration
19 μM) was added, followed by incubation for 24 h. After eachwell was washed twice with PBS, 100 μL of RIPA buffer was added
and the cells in the well were lysed. The cell lysate was moved to a
96-well plate, and the ﬂuorescence intensity was measured by amicroplate reader. The excitation and ﬂuorescence wavelengths
were 420 and 675 nm, respectively. Since the number of cells
cultured in each well differed, the ﬂuorescence intensity was
normalized by the number of cells in each well. Since the amount

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of total protein in each well is related to the number of cells, theamount of total protein in each well after ﬂuorescence measurement was analyzed using the BCA Protein Assay Kit (TaKaRa BioInc.) to normalize the PLP data.

Reporting summary. Further information on research design isavailable in the Nature Portfolio Reporting Summary linked tothis article.
Data availability

Endoplasmic reticulum and mitochondria staining. ERTracker™ Green (Invitrogen™) and MitoTracker™ Orange
CMTMRos (Invitrogen™) were used for ﬂuorescence staining of
the ER and mitochondria, respectively. PLP-added RGK1 and
RGM1 cells were cultured in a glass-bottom dish of 35 mm diameter and allowed to stand for 24 h in an incubator at 37 °C witha CO2 concentration of 5%. Next, 0.5 μL of ER Tracker DMSO
solution at 1 mM concentration and 0.5 μL of MitoTracker
DMSO solution at 1 mM concentration were added to 2 mL of
Hank’s balanced salt solution (GibcoTM, Thermo Fisher ScientiﬁcK.K.). Then this solution was replaced with the medium in thedish and allowed to stand for 10 min in an incubator. Finally, thestaining solution in the dish was replaced with the medium, andthe sample was observed using a super-resolution microscope.

Numerical source data for Fig. 3, Fig. 7, and Fig. 8 can be found in Supplementary Data 1,
Supplementary Data 2 and Supplementary Data 3, respectively.

Received: 1 September 2023; Accepted: 16 November 2023;

References
1.
2.
3.
4.

DAPGreen staining. DAPGreen (Dojindo Laboratories Co., Ltd.)detects autophagosomes and autolysosomes. DAPGreen isincorporated in autophagosomes during double membrane formation and emit ﬂuorescence under hydrophobic conditions.
The cells cultured in a glass-bottom dish shown in Fig. 5a, bwere allowed to stand in an incubator for 24 h after PLP dosing.
After washing the sample twice with a new medium without PLP,
2 mL of the medium was added and 2 μL of 0.1 mM DAPGreenDMSO solution was added. After being allowed to stand in the
incubator for 30 min, the medium in the dish was removed bywashing twice with a new medium without DAPGreen. Then,
2 mL of the medium was added, and the sample was observed
using a super-resolution microscope.
For the cells in Fig. 5b, d, the DAPGreen staining and PLP
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incubator for 30 min, the medium in the dish was removed,
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Acknowledgements
We acknowledge the ﬁnancial supports of a Grant-in-Aid for Scientiﬁc Research(23H00264) from Japan Society for the Promotion of Science and Japan Core Research
Evolutional Science and Technology (CREST) (JPMJCR1875).

Author contributions
A.T. developed the system and took and analyzed the microscopy-based data. H.K. and
M.K. performed the Western blot measurement and the measurement of PLP concentration in cells. O.T., Y.A., H.M., and T.O. provided technical advice. H.S. organized
and supervised the project and edited the paper with A.T.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s42003-023-05598-0.
Correspondence and requests for materials should be addressed to Hidemi Shigekawa.
Peer review information Communications Biology thanks Kazutaka Hirakawa and theother, anonymous, reviewer(s) for their contribution to the peer review of this work.
Primary Handling Editors: Gregory Lavieu and Johannes Stortz.
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