Laser-triggered intelligent drug delivery and anti-cancer photodynamic therapy using platelets as the vehicle

Abstract In our previous study, target drug delivery and treatment of malignant tumors have been achieved by using platelets as carriers loading nano-chemotherapeutic agents (ND-DOX). However, drug release from ND-DOX-loaded platelets is dependent on negative platelet activation by tumor cells, whose activation is not significant enough for the resulting drug release to take an effective anti-tumor effect. Exploring strategies to proactively manipulate the controlled release of drug-laden platelets is imperative. The present study innovatively revealed that photodynamic action can activate platelets in a spatiotemporally controlled manner. Consequently, based on the previous study, platelets were used to load iron oxide-polyglycerol-doxorubicin-chlorin e6 composites (IO-PG-DOX-Ce6), wherein the laser-triggered drug release ability and anti-tumor capability were demonstrated. The findings suggested that IO-PG-DOX-Ce6 could be stably loaded by platelets in high volume without any decrease in viability. Importantly and interestingly, drug-loaded platelets were significantly activated by laser irradiation, characterized by intracellular ROS accumulation and up-regulation of CD62p. Additionally, scanning electron microscopy (SEM) and hydrated particle size results also showed a significant aggregation response of laser irradiated-drug-loaded platelets. Further transmission electron microscopy (TEM) measurements indicated that the activated platelets released extracellularly their cargo drug after laser exposure, which could be taken up by co-cultured tumor cells. Finally, the co-culture model of drug-loaded platelets and tumor cells proved that laser-triggered delivery system of platelets could effectively damage the DNA and promote apoptosis of tumor cells. Overall, the present study discovers a drug-loaded platelets delivery using photodynamic effect, enabling laser-controlled intelligent drug delivery and anti-tumor therapy, which provides a novel and feasible approach for clinical application of cytopharmaceuticals. Graphic Abstract Plain Language Summaries What is the context? 1. Platelets were applied to load IO-PG-DOX-Ce6, wherein the laser-triggered drug release and anti-tumor effect were investigated in vitro. 2. The findings indicated that IO-PG-DOX-Ce6 could be stably loaded by platelets in high volume without any decrease in viability, which may attribute to the activation of autophagy in platelets. 3. IO-PG-DOX-Ce6-loaded platelets could be significantly activated by laser irradiation (690 nm). 4. Activated platelets released extracellularly their cargo drug after laser exposure, which could be taken up by co-cultured tumor cells 5. The co-culture model of drug-loaded platelets and tumor cells proved that the laser-triggered delivery system of platelets could effectively damage the DNA and promote apoptosis of tumor cells. What is new? 1. Platelets could be utilized as the vehicle to load photosensitizer-loaded-nano-drug. 2. Photodynamic action can activate platelets in a spatiotemporally controlled manner, which could be a tool to regulate the activation of platelets. 3. The laser-triggered activation of drug-loaded platelets allows for target release of cargo. 4. The limitation of the current research is that only in vitro experiments were carried out to demonstrate our conclusions. What is impact? The present work provides a novel and feasible approach for the clinical application of cytopharmaceuticals.


Introduction
Platelets are abundant, tiny, with long circulation effects, and thereby are excellent cellular carriers easily accessible to transport drugs to the neovascularization of tumors [1][2][3].Platelet activation leads to the adhesion to the vascular endothelium and releasing the internal granules [4][5][6][7][8], which is important for drug delivery [9,10].Consequently, controlled regulation of platelet activation holds potential for targeted drug delivery.Chlorin e6 (Ce6) is frequently used as a photosensitizing drug with low toxicity, fluorescent tracer, which could be activated by laser irradiation to generate reactive oxygen species (ROS) for photoselective cellular modulation or destroying [11,12].However, few studies have concerned the effect of photodynamic effects on platelets so far.There is evidence suggesting that oxidative stress can induce platelet activation [13][14][15][16].Thus, platelets loaded with Ce6 are presumed to be activated by laser irradiation to release the loaded photosensitizer, achieving laser-triggered drug delivery.
However, Ce6 has a large amount of negative surface charge and thereby is loaded in limited volume by platelets with negative membrane surface charge.In our previous study, Ce6 and Doxorubicin (DOX), a chemotherapeutic agent with pro-cell membrane properties, were co-linked to Iron Oxide (IO) nanocarriers modified with Polyglycerol (PG).A photodynamic nanoparticle IO-PG-DOX-Ce6 was prepared [17][18][19].IO-PG-DOX-Ce6 worked with both photodynamic therapy and chemotherapy, wherein the pro-cellular membrane properties from DOX can significantly improve the enrichment and stability of Ce6 in platelets.Additionally, immunomodulatory molecules on the platelet membrane surface (e.g.CD47) in turn could prevent the nanoparticle from being cleared by the immune system [20,21].Most critically, Ce6-mediated photodynamic effects could produce ROS to activate drug-loaded platelets in the presence of laser irradiation to achieve intelligent drug delivery in a light- Laser-activated platelets drug delivery and therapy 3 controlled manner.Hence, once the IO-PG-DOX-Ce6-loaded platelets are chemotactic to the cancer cells, laser irradiation allows for light-controlled drug delivery and targeted therapy.
To test this concept, the present work examined the capacity and stability of platelets loaded with IO-PG-DOX-Ce6.Then, we further verified the ways by which platelets take up and load this nanoparticle.Additional studies explored the activation of drug-laden platelets by laser irradiation and the ability of activated platelets to deliver the loaded drug to co-cultured tumor cells.Finally, laser irradiation-mediated targeted drug delivery and anti-tumor photodynamic efficacy were demonstrated in the co-culture system.In the present work, the concept of "laser-triggered activation of drug-loaded platelets" was proposed, whose potential application and drug-loading-related mechanisms were also proved using our prepared nanoparticle IO-PG-DOX-Ce6.

IO-PG-DOX-Ce6 conjugates
IO-PG-DOX-Ce6 was prepared according to our previous reports [17].Briefly, iron oxide (IO) nanoparticles with a surface coating of polyglycerol (IO-PG) and bis(p-nitrophenyl) carbonate were dissolved in DMF and reacted at 140°C for 24 h to yield IO-PG-PhNO 2 .IO-PG-PhNO 2 was reacted with hydrazine hydrate in DMF at 90°C for 12 h.The as-synthesized were purified and ultra-filtration centrifugation to afford IO-PG-NHNH 2 .Finally, IO-PG-NHNH 2 was ultrasonically dispersed with DOX into pure water with a pH of 10 and reacted overnight at 50°C.The products were purified to yield IO-PG-DOX.Finally, IO-PG-DOX was reacted with Ce6 under alkaline conditions at 37°C for 24 h to yield IO-PG-DOX-Ce6 (Fig. S1).The IO-PG-DOX-Ce6 solution was kept in pure water at 4°C.All concentrations and dosages of IO-PG-DOX-Ce6 were normalized to Ce6.

Platelets and cancer cells culture
Platelets were prepared according to our published protocols [23].Briefly, platelet-rich plasma was harvested from peripheral blood extracted from female C57BL mice (6-8 weeks of age, 22-24 g) under nembutal anesthesia.Blood is collected by anticoagulation tubes preloaded with sodium citrate to prevent activation of platelets after isolation.All platelet groups in the present study were treated with the same standard of anticoagulation to exclude the effect of anticoagulation tubes on the results.Animal handling and experimental procedures were in line with protocols approved by the Animal Care Committee at the Hubei University of Medicine.The platelet-rich plasma was further centrifuged at 100 g for 20 min for thorough removal of red blood cells.Then the supernatant was centrifuged at 800 g for 20 min to collect the platelets, which were identified by confocal microscopy (FV3000RS, Olympus) and TEM (HT7700, Hitachi, Japan) (Fig. S2).Lewis lung cancer cells (LLC) were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China).Cell cultures were conducted with DMEM medium added with 10% fetal bovine serum (QmSuero/Tsingmu Biotechnology,Wuhan) in a humidified incubator (5%CO 2 atmosphere at 37°C).

Drug uptake assay
To investigate the IO-PG-DOX-Ce6 loading in platelets, the IO-PG-DOX-Ce6 or Ce6-incubated platelets were harvested.The fluorescence of Ce6 in platelets was measured by flow cytometry or observed by high-content microscopy (Operetta CLS, Perkin Elmer).The endocytosis of platelets was inhibited by Pitstop (PT).Briefly, the platelets were treated with PT (30μM) for 1h before incubating with IO-PG-DOX-Ce6.Alternatively, Hydroxychloroquine (HCQ) with a concentration of 0.67mg/ml was utilized to block the platelets' autophagy.The fluorescence of Ce6 in platelets and cancer cells was detected by flow cytometry (cytoflex, Beckman Coulter, USA) respectively in the co-cultured system, wherein the platelets were pre-treated with HCQ before the addition of IO-PG-DOX-Ce6.

Autophagy, Viability assay of nanodrug-loaded platelets
The proteins from IO-PG-DOX-Ce6-loaded platelets were extracted and used to measure the expression of Beclin-1.Alternatively, the nanodrug-loaded platelets were harvested and fixed.The morphology of autophagosomes was observed by TEM.For detection of the cell viability, nanodrug-loaded platelets were seeded to 96-well plates and incubated with CCK-8 for 2-4h and then detected by a plate reader (SpectraMax i3, Molecular Devices), wherein the absorbance at 450nm was recorded.The proteins of CD42b and Caspase-3 from drug-loaded platelets were assayed by WB.In addition, platelets were harvested and stained by Rhodamine 123.The fluorescence of Rhodamine 123 was assayed with flow cytometry, wherein the decreased fluorescence reflected the loss of viability.Laser-activated platelets drug delivery and therapy 5 Laser-activated platelets drug delivery and therapy 7

Measurement of platelets' activation
First, the nanodrug-loaded platelets were obtained and labeled by DCFH-DA probe after the laser irradiation (690nm, 0.5 w, 60 s).The ROS generation was counted by the fluorescence of DCF.And then the membrane expression of CD62p in drug-loaded platelets was assayed by flow cytometry.The activated drugloaded platelets were observed with SEM after fixed on a slide.Additionally, the hydrated particle size of IO-PG-DOX-Ce6loaded platelets was assayed by a dynamic light scattering (DLS).

Co-culture of IO-PG-DOX-Ce6-loaded platelets with cancer cells
IO-PG-DOX-Ce6-loaded platelets were co-cultured with Lewis cells for 6h.The co-culture was then irradiated by laser.The Ce6 fluorescence of platelets and cancer cells was assayed by flow cytometry respectively.Based on this, a second laser irradiation was conducted, which was 1h away from the first irradiation.Then the suspended platelets were removed.The cancer cells were harvested and labeled by DAFH-DA probe for detecting of the ROS production.Alternatively, the collected cancer cells were stained by Annexin-V for measurement of the apoptosis level.Furthermore, cancer cells were utilized for comet analysis.The proteins from cancer cells were also extracted for apoptosisassociated molecules assay.

Anti-cancer effects in vitro
To examine the anti-tumor effects, the cancer cells in co-culture system of platelets and cancer cells were collected and then labeled by DCFH-DA probe.The ROS generation was counted by DCF fluorescence.Alternatively, the DNA damage and  apoptosis-associated molecules extracted from cancer cells, such as p53, Bax and Caspase-3, were detected using WB.The comet assay was applied to detect the DNA double-strand breakage.The harvested cancer cells were stained with Annexin-V (Purchased from CHAMOT BIOTECHNOLOGY CO., LTD.) and then assayed with flow cytometry.

Flow cytometry, Microscopy and Western blotting (WB)
Drug fluorescence was measured with flow cytometry.Ce6 fluorescence was acquired in the APC channel.DOX fluorescence was acquired in the PE channel.At least 1×10 4 cells were acquired for every collection.Geometric means (GM) were used to quantify the mean fluorescent intensity (MFI).To observe the drug distribution in platelets, the nanodrug-loaded platelets were photographed by confocal microscopy, high content minor, TEM or SEM.For WB assay, drug-loaded platelets were rinsed twice and then lysed.Cell lysates were centrifuged and quantified with a BCA assay kit.The same protein aliquots were fractionated by SDS-PAGE and transferred to PVDF membranes.The membranes were blocked with 4% bovine serum albumin in TBST and incubated with antibodies including CD42b (11026-1-AP, Proteintech, Wuhan, China), Caspase-3 (50599-2-Ig, Proteintech, Wuhan, China), Beclin-1 (bs-1353R, Bioss, Beijing, China), p53 (bs-2090R, Bioss, Beijing, China), Bax (bs-0127R, Bioss, Beijing, China) and GADPH (PMK053C, BioPM, Wuhan, China) at 4°C overnight.The membranes were washed and then incubated with horseradish peroxidase-conjugated secondary antibody before developing with a ECL kit (PMK003, BioPM, Wuhan, China).

Statistical analysis
The measured data in this article were displayed using the mean ± standard deviation (SD).Statistical differences among groups were assayed by One-way analysis of variance (ANOVA).Tukey test was utilized for the correct multiple comparisons.P values <0.05 was recognized to be statistically significant.

Platelets could be utilized as an efficient carrier for IO-PG-DOX-Ce6 loading
As mentioned, in the conjugates, DOX, a chemotherapeutic agent with pro-cell membrane properties, was co-linked to Iron Oxide (IO) nanocarriers modified with Polyglycerol (PG), which could significantly improve the enrichment and stability of Ce6 in platelets.As expected, IO-PG-DOX-Ce6 could be loaded by platelets with a high capacity compared with the treatment of Ce6 at the same concentration (2μg/ml of Ce6), as confirmed by enhanced fluorescence of Ce6 in platelets (Figure 1a,b).Additionally, high-content microscopy detection identified that IO-PG-DOX-Ce6-loaded platelets held the IO-PG-DOX-Ce6 for at least 10h in a fresh culture medium without release (Figure 1c).Further analysis showed that endocytosis inhibitor pitstop (PT) treatment effectively wakened the IO-PG-DOX-Ce6 accumulation in platelets (Figure 1d,e), indicating that platelets may take up IO-PG-DOX-Ce6 nanoparticle mainly by endocytosis.This proof revealed platelets could be applied as an efficient vehicle for IO-PG-DOX-Ce6 loading and delivery.Laser-activated platelets drug delivery and therapy 9 The loading of IO-PG-DOX-Ce6 by platelets was in an autophagy-dependent model Nanosized agents are typically engulfed by autophagosomes after swallowed by cells to prevent disruption of cellular homeostasis.To test whether a similar pattern is present in platelets, the autophagy of platelets was investigated.As displayed in Figure 2a-c, the IO-PG-DOX-Ce6 triggered significant autophagy of platelets, as characterized by enhanced expression of Beclin-1 and observation of double-layered membrane structure of autophagosomes.Consistently, autophagy inhibitor pre-treatment suppressed the loading of IO-PG-DOX-Ce6 in platelets (Figure 2d-f), further evidencing the loading of IO-PG-DOX-Ce6 by platelets was in an autophagy-dependent model.At the same time, the cell viability of IO-PG-DOX-Ce6-loaded platelets varied little measured by CCK-8 (Figure 3a).The expression of CD42b (a biomarker of platelets) and cleaved-caspase-3 was unchanged in the presence of IO-PG-DOX-Ce6 (Figure 3b-d).The fluorescence of Rhodamine 123 did not decrease in IO-PG-DOX-Ce6-loaded platelets (Figure 3e-f).These findings suggested that IO-PG-DOX-Ce6-treated platelets maintained excellent viability, which was a prerequisite for subsequent laser activation of IO-PG-DOX-Ce6-loaded platelets.

Laser irradiation encouraged nanodrug-loaded platelets to release IO-PG-DOX-Ce6 into tumor cells
For a demonstration of the laser-triggered activation of IO-PG-DOX-Ce6 loaded platelets, intracellular ROS generation was detected first.The production of ROS was elevated in nanodrugloaded platelets in response to laser irradiation (690nm, 0.5 w, 60s) as presented in Figure 4a-b, which was assayed by the DCFH-DA probe.On top of that, the results of membrane biomarkers in drug-loaded platelets evidenced that laser irradiation up-regulated the expression of CD62p when platelets carried photosensitizers (Figure 4c-e and Fig. S3).Moreover, the prominent aggregation of drug-loaded platelets was observed in the presence of laser irradiation (Figure 4f).The findings of hydrated particle size also identified laser irradiation could activate IO-PG-DOX-Ce6-loaded platelets, as characterized by the increased particle size (Figure 4g).In summary, laser irradiation could activate IO-PG-DOX-Ce6-loaded platelets.
Depending on the release reaction of activated platelets, the release of the IO-PG-DOX-Ce6 is monitored in the following experiments.As presented in Figure 5a, the extracellular IO-PG-DOX-Ce6 particles in large quantities can be visualized by TEM in response to laser irradiation.The released IO-PG-DOX-Ce6 could be taken up by co-cultured cancer cells, as confirmed by the strengthened fluorescence of Ce6 and DOX in cancer cells (Figure 5b).In agreement with the TEM and confocal microscopy, the results of flow cytometry also revealed that laser irradiation promoted the drug release from IO-PG-DOX-Ce6-loaded platelets and drug uptake into cancer cells (Figure 5c-f).Interestingly, Hydroxychloroquine (HCQ) treatment effectively reduced the uptake of Ce6 in cancer cells, indicating the release of nanodrug from platelets may be in an autophagydependent model as well (Figure 5g-h).Taken together, laser irradiation encouraged nanodrug-loaded platelets to release IO-PG-DOX-Ce6 into tumor cells.This laser-triggered release of drugloaded platelets enabled photo targeting of drug delivery with intelligent features.

Multiple laser irradiation induced significant DNA damage and apoptosis in tumor cells containing IO-PG-DOX-Ce6
Finally, massive ROS generation could be measured in cancer cells of co-culture system by a second time of laser irradiation, which was 1h away from the first irradiation (Figure 6a-c).In addition, pronounced DNA damage along with significant apoptosis also be detected in cancer cells, as characterized by increased expression of p53, cleaved-caspase-3 and Bax (Figure 6d-g), obvious tail of comet (Figure 6h-i), intensified Annexin-V-positive cells (Figure 6j-k).These data were crucial evidences that multiple laser irradiation induced significant DNA damage and apoptosis in tumor cells containing IO-PG-DOX-Ce6.

Discussion and conclusion
The current research explored the possibility and practicality of platelets-mediated tumor delivery of photosensitizer with lasertriggered drug release property, wherein the multiple laser irradiation could further induce significant apoptosis of malignant cells (Figure 7).
Clinically, many malignant tumors, including lung cancer and breast cancer, have high recurrence rates, which mainly originate from the difficulty of complete removal of infiltrating growing malignant cells.Conventional administration can not better enrich to the deep blood vessels of cancer tissues, leading to treatment failure.Previous work has primarily emphasized the development of different photodynamic-chemotherapy nanopharmaceutical systems, including IO-PG-DOX-Ce6, IO-PG-Glu-Ce6, and ND-PG-DOX [17][18][19].These nanoagents have excellent therapeutic effect and directly destroy malignant cells.However, these nanocomposites (e.g.IO-PG-DOX-Ce6) do not have controlled release ability.Therefore, we have previously used nanoparticle to demonstrate tumor drug delivery mediated by immunocytes and platelets [19,[22][23][24].On the one hand, platelets can enrich to the neovascularization of tumor tissues with loaded drug and are important cells for regulating vascular function and blood clotting.On the other hand, IO-PG-DOX-Ce6 enhanced the captivity and stability of photosensitizers within platelets.Photodynamic action to modulate platelet function is rarely reported.This "laser-controlled activation" proposed in the current study is significant in that the photodynamic effect can be used as a temporally and spatially controlled means of activation (activated on time, at specific tissue sites), whose activation effect can further be utilized as a tool of targeted drug release and vascular blockage.This realizes the purpose of selective modulation of platelet function for the treatment of diseases.In addition, replacement or addition of the corresponding drug or ligand in the nanoparticle can be of great value for targeted intervention in other diseases.
This study also revealed that IO-PG-DOX-Ce6 could evoke platelet autophagy, which may be temporarily stored in autophagosomes.Autophagy is essential for the maintenance of platelet function [25][26][27][28], wherein initiation of autophagy isolates foreign particles from the autophagosome to ensure that the normal function of platelets is not disturbed.We thus demonstrated that platelets encapsulate IO-PG-DOX-Ce6 in intracellular autophagosomes via cytocytosis and further release the drug extracellularly via the autophagic lysosomal pathway.In addition to photodynamic action, other techniques that can temporally and spatially modulate platelet activation can also be applied for smart drug delivery.For instance, photothermal action has been discovered to trigger the activation of platelets to form thrombi locally in tumor vessels and continuously release loaded agents [29].Besides, other cellular components in blood, such as red blood cells and neutrophils, are also excellent carriers for long-acting or penetrating targeted drug delivery [30,31].
In summary, the present work discloses a drug-loaded platelets delivery using photodynamic effect, allowing laser-induced smart drug delivery and anti-tumor therapy in vitro, which provides a novel and feasible approach for clinical application of cytopharmaceuticals.

Figure 1 .
Figure 1.IO-PG-DOX-Ce6 could be taken up by platelets in a high volume stably.a-b: the fluorescence of IO-PG-DOX-Ce6 or Ce6 in platelets was detected using flow cytometry.c: Time-lapse observation of platelets loaded with IO-PG-DOX-Ce6 or Ce6.The fluorescence was detected with high-content screening.d-e: the uptake of drug was blocked by endocytosis inhibitor (pitstop, PT).The fluorescence of IO-PG-DOX-Ce6 was measured by flow cytometry.Geometric means were applied to count the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p < 0.05).Note: PT referred to Pitstop treatment.

Figure 2 .
Figure 2. IO-PG-DOX-Ce6 induced autophagy in platelets.a-b: The expression of Beclin-1 was detected by WB.The mean gray of bands was quantitatively analyzed.c: The autophagosomes in IO-PG-DOX-Ce6-loaded platelets were observed with TEM.Autophagosomes were marker by yellow arrow.d-f: The intracellular fluorescence of drug was measured by flow cytometry (d-e) and confocal microscopy (f) in the presence of HCQ.Geometric means were applied to count the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p < 0.05).Note: ID6 refers to IO-PG-DOX-Ce6.

Figure 3 .
Figure 3. IO-PG-DOX-Ce6-loaded platelets maintained viability.a: the cell viability of nanodrug-loaded platelets was assayed by CCK-8.b-d: the expression of CD42b and Caspase-3 in platelets was measured with WB.The mean gray of bands was quantitatively analyzed.e-f: the Rhodamine 123 was applied to assay the cell viability of platelets.The fluorescence of Rhodamine 123 was assayed by flow cytometry.Geometric means were applied to count the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p < 0.05).

Figure 4 .
Figure 4. Laser irradiation activated IO-PG-DOX-Ce6-loaded platelets.a-b: the ROS generation in nanodrug-loaded platelets under laser irradiation was assayed by DCFH-DA staining.c-e: Flow cytometry revealed the membrane expression of p-selectin (Cd62p).f: the aggregation of nanodrug-loaded platelets was observed using SEM.g: The hydrated particle size of platelets was assayed with DLS.Geometric means were used to quantify the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p <0 .05).Note: (L) refers to laser irradiation treatment.

Figure 6 .
Figure 6.IO-PG-DOX-Ce6 taken up by tumor cells elicited prominent oxidative DNA damage and apoptosis with multiple laser irradiation.a: the diagrammatic sketch of laser-triggered drug release and anti cancer effect with nanodrug-loaded platelets in the present work.b-c: the ROS production in tumor cells was assayed by DCFH-DA probe.d-g: the DNA damage and apoptosis-associated molecules (p53, Bax and Caspase-3) in tumor cells were measured by WB.The mean gray of bands was quantitatively assayed.h-i: Comet experiments investigated the DNA double-strand breakage of tumor cells.j-k: the apoptosis of tumor cells was evaluated through Annexin-V/PI staining.The Annexin-V-positive cells were counted.Geometric means were used to quantify the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p <0 .05).Note: (1 st LI) refers to laser irradiation treatment once in co-culture system.(1 st +2 nd LI) refers to laser irradiation treatment two times in co-culture system.

Figure 5 .
Figure 5.The activated nanodrug-loaded platelets deliver IO-PG-DOX-Ce6 into tumor cells.a: TEM observation of IO-PG-DOX-Ce6 release in laser-treated nanodrug-loaded platelets.b: the cellular fluorescence of nanodrug in tumor cells was photographed by confocal microscopy.c-f: the coculture model of nanodrug-loaded platelets and tumor cells was conducted under laser irradiation, wherein the drug release of platelets and drug uptake of tumor cells was assayed by flow cytometry respectively.g-h: Geometric means were used to quantify the mean fluorescence intensity (MFI) of flow cytometry.Values were means ± SD (n = 3, *p <0 .05).Note: (L) refers to laser irradiation treatment.

Figure 7 .
Figure 7.The schematic diagram of the present study.Laser at responsive wavelengths activates IO-PG-DOX-Ce6-loaded platelets, which in turn facilitates platelets' spatiotemporally controlled release of drugs.Re-laser irradiation after drug uptake by tumor cells can trigger photodynamic effects, thus destroying malignant cells.