Self-Assembled Nanocomposite DOX/TPOR4@CB[7]4 for Enhanced Synergistic Photodynamic Therapy and Chemotherapy in Neuroblastoma

DOX/TPOR4@CB[7]4 was synthesized via self-assembly, and its physicochemical properties and ability to generate reactive oxygen species (ROS) were evaluated. The impact of photodynamic therapy on SH-SY5Y cells was assessed using the MTT assay, while flow cytometry analysis was employed to detect cell apoptosis. Confocal laser scanning microscopy was utilized to observe the intracellular distribution of DOX/TPOR4@CB[7]4 in SH-SY5Y cells. Additionally, fluorescence imaging of DOX/TPOR4@CB[7]4 in nude mice bearing SH-SY5Y tumors and examination of the combined effects of photodynamic and chemical therapies were conducted. The incorporation of CB[7] significantly enhanced the optical properties of DOX/TPOR4@CB[7]4, resulting in increased ROS production and pronounced toxicity towards SH-SY5Y cells. Moreover, both the apoptotic and mortality rates exhibited significant elevation. In vivo experiments demonstrated that tumor growth inhibition was most prominent in the DOX/TPOR4@CB[7]4 group. π–π interactions facilitated the binding between DOX and photosensitizer TPOR, with TPOR’s naphthalene hydrophilic groups encapsulated within CB[7]’s cavity through host–guest interactions with CB[7]. Therefore, CB[7] can serve as a nanocarrier to enhance the combined application of chemical therapy and photodynamic therapy, thereby significantly improving treatment efficacy against neuroblastoma tumors.


Introduction
Neuroblastoma (NB) is an embryonic tumor of the sympathetic system, originating from 8% to 10% of tumors in children.It represents the most prevalent malignant solid neoplasm in infants and young children, ranking as the second most common extracranial malignancy among this age group [1][2][3].Neuroblastoma displays remarkable phenotypic heterogeneity [4].High-risk patients have a dismal prognosis, with a survival rate below 50%, even after undergoing chemotherapy, radiotherapy, and surgery [5,6].Consequently, there is an urgent need to develop innovative and reliable therapeutic strategies for enhancing neuroblastoma treatment.
Due to the multidrug resistance exhibited by tumor cells and the inherent heterogeneity of tumors, monotherapy alone is insufficient for the complete eradication of tumors.A complementary strategy involving the combination of multiple therapies with distinct mechanisms is imperative [7][8][9].Photodynamic therapy (PDT) is a clinically approved noninvasive therapeutic modality of significant importance [10].PDT is regarded as an optimal noninvasive modality that offers precise tumor treatment with simplicity, flexibility,

Chemical Synthesis and Characterization
2.1.1.Preparation of DOX/TPOR 4 @CB [7] 4 The TPOR, DOX, and CB [7] were individually dissolved in DMSO and subjected to 4 h of ultrasonication under light-avoidance conditions.The TPOR and DOX were individually stirred for 12 h under conditions of light avoidance.The DOX solution was gradually introduced into the TPOR solution and agitated for 24 h under light-avoidance conditions to obtain the DOX/TPOR 4 solution, then added into the solution of CB [7] in definite proportion with 1:4.Characterization of ultraviolet-visible spectra and fluorescence spectra were detected by ultraviolet spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan) and fluorescence spectroscopy (Cary eclipse, VRIAN, Santa Clara, CA, USA).

Morphological Characteristics
DOX/TPOR 4 @CB [7] 4 was synthesized in the same way, with methanol as the solvent.It was then dried by a freeze-drying machine to form a solid powder for scanning electron microscope (SEM NANO450, FEI) and transmission electron microscope (TEM JEM2100F, Tokyo, Japan) observation.

Cells and Animals
Neuroblastoma cells SH-SY5Y was obtained from the American Type Culture Collection (ATCC) and cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Every Green, Shenzhen, China), and 1% penicillin-streptomycin (Gibco, USA).Cells were maintained at 37 • C in a 5% CO 2 incubator.Healthy female BALB/c-nude mice (4-6 weeks old) were purchased from SPF (Beijing, China) Biotechnology Co, Ltd.Tumor-bearing mice were constructed by subcutaneously injecting a suspension of 1 × 10 7 SH-SY5Y cells.All mice procedures were approved by and raised in the SPF laboratory animal room.

Intracellular ROS Detection
To detect the intracellular ROS, we used the 2,7,-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) as a probe.Collected cells were inoculated into confocal culture dishes (φ15 mm glass dish, 8 × 10 4 cells/dish) and cultured for attachment at 37 • C in 5% CO 2 incubator.Then, incubated with 2.5 µM TPOR, DOX/TPOR 4 , DOX/TPOR 4 @CB [7] 4 and 0.625 µM of DOX for 24 h.Cells were washed three times, and DCFH-DA (10 µM) was added and incubated for 30 min.525 nm laser was used to illuminate with 95.5 mW/cm 2 for different periods.The fluorescence images of the cells were observed by the Confocal laser scanning microscope (CLSM, Nikon, Kyoto, Japan).The mean density was analyzed by Image-Pro Plus 6.0.

Drug Cellular Localization Test
The cells were seeded in confocal culture dishes (φ15 mm glass dish) and cultured for attachment.The cells were incubated with 2.5 µM TPOR, DOX/TPOR 4 , DOX/TPOR 4 @CB [7] 4 , and 0.625 µM DOX for 24 h.After being washed with PBS three times, DAPI (Solarbio, Beijing, China) was added.After incubation for 30 min, it was washed with PBS for five times.After incubation with MitoTracker Green for 45 min, it was washed with PBS for three times and imaged with CLSM.

In Vitro Synergistic Therapeutic Effects of PDT and Chemotherapy
The cells were incubated with 0.625 µM DOX and 2.5 µM CB [7], TPOR, DOX/TPOR 4 , DOX/TPOR 4 @CB [7] 4 for 24 h.Then, the cells were replaced with fresh DMEM medium and irradiated with 525 nm laser for 0.5 min, 1 min, 2 min and 3 min at 95.5 mW/cm 2 intensity in the dark room.The survival rate of the cells was measured by MTT assay.

Flow Cytometry
Cell death was assessed post-PDT using the Apoptosis Assay Kit (KeyGEN, Nanjing, China).Treated and control samples were analyzed using FlowJo VX on the BD FACS Celesta (BD, San Jose, CA, USA).

In Vivo Targeting Ability Evaluation
The in vivo tumor-targeting ability of DOX/TPOR 4 @CB [7] 4 was evaluated by fluorescent imaging on an IVIS Spectrum system (PerkinElmer, Waltham, MA, USA).Briefly, 2.5 µM DOX and 10 µM TPOR DOX/TPOR 4 and DOX/TPOR 4 @CB [7] 4 were intravenously injected into the tumor bearing mice and the fluorescent images of mice at intervals of 0 h, 12 h, 24 h, 36 h, and 48 h were observed and recorded.

In Vivo Anti-Tumor Evaluation
The tumor-bearing mice were randomly divided into four groups (n = 6 mice/group) with treatments of PBS + Light, TPOR + Light, DOX/TPOR 4 + Light, and DOX/TPOR 4 @CB [7] 4 + Light.These mice were injected for drug administration and subsequently exposed to darkness in a controlled environment, then received photodynamic therapy after 24 h (6 min, 150 mW/cm 2 ).Every 3 days, drugs were injected to different treatments and received light irradiation after 24 h of intravenous injection according to the in vivo imaging results.Changes in tumor volume and mice body weight were recorded as well.After 18 days of treatment, the mice were euthanized and the harvested tumor tissues were fixed with 10% neutral formalin solution, followed by H&E staining and TUNEL staining analysis.

Statistical Analysis
All experiments were performed at least three times.Analysis was performed using SPSS Statistics 25.0.The one-way ANOVA test was used to compare the differences between multiple groups with significance accepted at p < 0.05.
We then discussed the UV absorption spectra and fluorescence spectra of the different ratios of DOX, TPOR and CB [7].We found that CB [7] had almost no absorption band in the UV-vis spectral range we were interested in.We observed a pronounced absorption band in the UV-vis spectral range of 500-700 nm for DOX/TPOR 4 -CB [7] 4 .Additionally, it exhibited intense fluorescence emission within the range of 600-800 nm (Figure S1) and the MTT assay demonstrated that photodynamic therapy exhibited the most significant efficacy (Figure S2), so DOX/TPOR 4 -CB [7] 4 was used in subsequent experiments and named DOX/TPOR 4 @CB [7] 4 .The corresponding UV-vis absorption spectrum is shown in Figure 1C.DOX had no obvious visible absorption in the range of 500-700 nm.We used the concentration of TPOR (10 µmol/L) as the basis to measure UV-vis absorption intensity.The concentration of TPOR (10 µmol/L) was utilized as the reference for measuring UV-vis absorption intensity; the absorption intensity of DOX/TPOR 4 was significantly lower than that of TPOR within the range of 600-800 nm.In addition, the absorption intensity of DOX/TPOR 4 @CB [7] 4 was significantly increased and strongest of three compounds (A TPOR = 0.4004, A DOX/TPOR4 = 0.2345, A DOX/TPOR4@CB [7] = 0.4248).The UV-vis absorption spectra data of DOX/TPOR 4 and DOX/TPOR 4 @CB [7] 4 revealed a slight blueshift (1-2 nm) in the absorption wavelength compared with TPOR.When excited at 520 nm, the fluorescence intensity of DOX/TPOR 4 decreased significantly compared with TPOR, and there was fluorescence quenching.
Moreover, the NH 2 group of DOX has a positive charge, which could be combined with the negatively charged Br group of TPOR through electrostatic action to enhance the interaction relationship [43].The fluorescence intensity of DOX/TPOR 4 @CB [7] 4 was relatively enhanced compared with DOX/TPOR 4 .TPOR and CB [7] can be combined by self-assembled, and there was a strong interaction between TPOR and CB [7], which can form a stable host-guest complex [41].The hydrophobic group of TPOR was fixed in the CB [7] cavity through host-guest interaction.The base nucleus of the porphyrin was large and remained outside the CB [7], which could still maintain the high concentration of porphyrin chromophore after combining with CB [7].Combined with the previous results, it was preliminarily proved that the self-assembled DOX/TPOR 4 @CB [7] 4 was formed [41].It suggested that CB [7] could enhance fluorescence intensity.Therefore, high fluorescence emission was achieved in the DOX/TPOR 4 @CB [7] 4 self-assembled nanosystem, which was conducive to observe in the cells and helpful for the visualization diagnosis and treatment of tumors.
After the addition of CB [7], the naphthalene hydrophobic groups of porphyrins in the compound were immobilized within the CB [7] cavity through host-guest interactions.The DOX/TPOR 4 @CB [7] 4 assembly also exhibited a lamellar stacking morphology with a more densely packed arrangement and reduced surface irregularities, indicating successful formation between CB [7] and DOX/TPOR 4 (Figure 1A).Size distribution analysis revealed that DOX/TPOR 4 @CB [7] 4 was predominantly concentrated in the range of 100-200 nm, with an average particle size of 120.9 nm (Figure 1B).Furthermore, it can be observed that among these three entities, DOX/TPOR 4 @CB [7] 4 displayed the highest Zeta potential (Figure S3B), with an average potential of 37.51 mV, demonstrating its relative stability.It was confirmed that DOX/TPOR 4 @CB [7] 4 possessed superior physical and chemical properties.However, with CB [7] being water-soluble, it provides a hydrophobic cavity while having a hydrophilic exterior that offers numerous opportunities to enhance photosensitizer properties [44].The further increase in particle size and zeta potential suggests successful loading of DOX/TPOR 4 with CB [7], effectively preventing TPOR self-aggregation.
ceutics 2024, 16, x FOR PEER REVIEW 6 nm, with an average particle size of 120.9 nm (Figure 1B).Furthermore, it can be obse that among these three entities, DOX/TPOR4@CB [7]4 displayed the highest Zeta pote (Figure S3B), with an average potential of 37.51 mV, demonstrating its relative stabil was confirmed that DOX/TPOR4@CB [7]4 possessed superior physical and chemical p erties.However, with CB [7] being water-soluble, it provides a hydrophobic cavity w having a hydrophilic exterior that offers numerous opportunities to enhance photos tizer properties [44].The further increase in particle size and zeta potential suggests cessful loading of DOX/TPOR4 with CB [7], effectively preventing TPOR self-aggrega

Revealing the Presence of Singlet Oxygen
In photodynamic effects, 1 O2 plays a crucial role in inducing tumor cell death generation of 1 O2 can be quantified by measuring its fluorescence intensity using a S fluorescence probe.As illustrated in Figure 1E, the time-dependent SOSG fluores intensity of all four drugs demonstrates their ability to produce a certain amount o Notably, when TPOR is combined with DOX, there is a significant enhancement in S fluorescence prior to binding.Previous studies have reported that DOX sensitizes p sensitizer-mediated photodynamic therapy [45,46].Consequently, DOX promote production of 1 O2 when used in conjunction with TPOR.Among the tested sam DOX/TPOR4@CB [7]4 exhibits the highest SOSG fluorescence intensity.After irradiatio 180s, the fluorescence intensity of DOX/TPOR4@CB [7]4 reaches as high as 600 unit proximately twice that of DOX/TPOR4 and six times that of TPOR alone.The additi

Revealing the Presence of Singlet Oxygen
In photodynamic effects, 1 O 2 plays a crucial role in inducing tumor cell death.The generation of 1 O 2 can be quantified by measuring its fluorescence intensity using a SOSG fluorescence probe.As illustrated in Figure 1E, the time-dependent SOSG fluorescence intensity of all four drugs demonstrates their ability to produce a certain amount of 1 O 2 .Notably, when TPOR is combined with DOX, there is a significant enhancement in SOSG fluorescence prior to binding.Previous studies have reported that DOX sensitizes photosensitizer-mediated photodynamic therapy [45,46].Consequently, DOX promotes the production of 1 O 2 when used in conjunction with TPOR.Among the tested samples, DOX/TPOR 4 @CB [7] 4 exhibits the highest SOSG fluorescence intensity.After irradiation for 180s, the fluorescence intensity of DOX/TPOR 4 @CB [7] 4 reaches as high as 600 units-approximately twice that of DOX/TPOR 4 and six times that of TPOR alone.The addition of CB [7] significantly influences the production and output of 1 O 2 .This discovery provides a robust foundation for potential applications in photodynamic therapy.

Reactive Oxygen Species(ROS) Quantification
Reactive oxygen species (ROS) play a key role in the photodynamic clearance of tumor cells [47].DCFH-DA fluorescent probe was used to detect ROS, which oxidized to DCF and emitted green fluorescence [48].As shown in Figure 2A, fluorescence was not observed in both the control group and the DOX group, indicating no ROS production.Under no light radiation, both DOX/TPOR 4 and DOX/TPOR 4 @CB [7] 4 exhibited obvious green fluorescence, and the fluorescence intensity of the latter was stronger.These experimental results in solution showed that DOX/TPOR 4 produced a certain level of ROS (Figure 1E).Furthermore, TPOR can be activated to produce ROS even when exposed to a short period of low-dose light under a microscope.DOX/TPOR 4 also has a stronger fluorescence intensity than TPOR alone under the same radiation conditions due to its ability to sensitize ROS.It is worth noting that among all test groups, DOX/TPOR 4 @CB [7] 4 demonstrated enhanced photosensitivity and resulted in a significantly augmented cellular drug uptake by SH-SY5Y cells.Moreover, in line with the findings of the 1 O 2 assay, the inclusion of CB [7] led to a further enhancement in intracellular ROS generation.These findings suggest that treatment with DOX/TPOR 4 @CB [7] 4 will have a more significant impact on tumor cell survival.Based on the host-guest interaction with CB [7], self-assembled compounds can effectively disperse and reduce TPOR accumulation under light excitation while producing ROS, which will have a significant tumor-killing effect [49,50].Experimental results demonstrate that self-assembled DOX/TPOR 4 @CB [7] 4 exhibits significantly enhanced optical properties, ROS yield, and improved drug stability.This suggests that CB [7] can enhance the fluorescence quantum yield of photosensitizers and contribute to their stability.

DOX/TPOR 4 @CB[7] 4 Co-Locates in Mitochondria
The subcellular localization of DOX/TPOR 4 @CB [7] 4 in SH-SY5Y cells was assessed by co-incubating DAPI as a nuclear stain and MitoTracker green as a mitochondrial marker for colocalization experiments.The emission fluorescence at 600-750 nm, corresponding to the inherent fluorescence properties of TPOR, was utilized to determine its cellular distribution.As shown in Figure 2C, TPOR, DOX/TPOR 4 , and DOX/TPOR 4 @CB [7] 4 all exhibited predominantly cytoplasmic red fluorescence.The fluorescence intensity of DOX/TPOR 4 was higher than that of TPOR due to the combination resulting in superimposed signals.Furthermore, the fluorescence intensity of DOX/TPOR 4 @CB [7] 4 was significantly enhanced compared to that of DOX/TPOR 4 and it displayed strong accumulation within SH-SY5Y cells.These findings suggest that DOX/TPOR 4 @CB [7] 4 , possessing suitable size and biocompatibility characteristics, efficiently entered the cells owing to TPOR's specific affinity [51,52].This indicates that under similar conditions, the addition of CB [7] improved the solubility of DOX/TPOR 4 and facilitated drug uptake by SH-SY5Y cells.Moreover, from the Figure 2C, it can be observed that red fluorescence emitted by DOX/TPOR 4 @CB [7] 4 showed good colocalization with MitoTracker green dye indicating a similar distribution pattern between drug molecules and mitochondria suggesting potential drug action on mitochondria.Under excitation by a 520 nm laser, DOX/TPOR 4 @CB [7] 4 emits bright fluorescence in the range of 600-750 nm, which falls within the near-infrared region.This property facilitates tissue and cell penetration as well as easy observation within cells.The increased fluorescence emission in the combination group aids in visualizing tumor diagnosis and treatment.
Experimental results demonstrate that self-assembled DOX/TPOR4@CB [7]4 exhibits significantly enhanced optical properties, ROS yield, and improved drug stability.This suggests that CB [7] can enhance the fluorescence quantum yield of photosensitizers and contribute to their stability.

Assessment of Phototoxicity, Dark Toxicity, and Potential Synergistic Effects
Previous experiments have demonstrated that CB [7] does not exert a significant inhibitory effect on the growth of SH-SY5Y cells within the concentration range of 1-10 µM [41].As depicted in Figure S4, laser radiation employed in this study did not induce any notable inhibition on cell viability within the range of 0~250 mW/cm 2 , thereby excluding the influence of light radiation on cell survival.In the absence of light radiation, the cell survival rate was lower in the DOX/TPOR 4 group compared to that in the DOX group at all concentrations, indicating that the chemotherapy drug DOX primarily contributed to cell death.The co-administration of DOX with TPOR enhanced DOX uptake, resulting in a more potent chemotherapy effect.Upon treatment with 1 µM DOX/TPOR 4 @CB [7] 4 , cellular viability rapidly decreased to approximately 74%, while significantly reducing the dosage of DOX as compared to the 10 µM DOX treatment group.Among all experimental groups, DOX/TPOR 4 @CB [7] 4 exhibited cytotoxicity and further augmented its (DOX) efficacy as a chemotherapeutic agent.The inclusion of CB [7] substantially improved medication effectiveness.We propose that the co-administration of DOX and TPOR drugs can enhance the intracellular uptake of DOX in tumor cells due to the unique affinity exhibited by photosensitizer TPOR, thereby eliciting potentiated chemotherapeutic effects.As depicted in Figure 3B, the results demonstrated that laser irradiation did not significantly impact the efficacy of DOX, Under laser radiation, the experimental group containing TPOR exhibited a significant decrease in cell survival rate with an evident time-dependent effect on cell killing.After 30 s of laser irradiation, cell viability in the DOX/TPOR 4 @CB [7] 4 group was approximately 33% (DOX: 84%, TPOR: 99%, DOX/TPOR 4 : 83%), indicating that DOX/TPOR 4 @CB [7] 4 exhibited superior synergistic effects for photodynamic and chemotherapy in vitro.The combination drug exhibits significant potential in reducing the dosage of chemotherapy, thereby effectively mitigating systemic toxicity and treatment-related side effects, offering promising advantages for neuroblastoma treatment.The synergistic application of photodynamic therapy and chemotherapy can mutually enhance each other's efficacy, surpassing the photodynamic effect observed in either TPOR group alone or DOX/TPOR 4 group alone.

The Analysis of Flow Cytometry
To further assess the anti-tumor efficacy of the drug, we employed the ANNEXIN V-FITC/PI double labeling method to quantify the ratio of apoptosis to necrosis following PDT treatment [53].As depicted in Figure 3C, in the absence of laser irradiation, no significant differences were observed in apoptosis compared to the control group.However, 4 h after 2 min of irradiation, both the DOX/TPOR 4 group and DOX/TPOR 4 @CB [7] 4 group exhibited significant rates of apoptosis at 20.93% and 28.0%, respectively.It is noteworthy that flow cytometry analysis of DOX/TPOR 4 @CB [7] 4 yielded consistent results with the MTT assay, demonstrating pronounced cytotoxic effects on SH-SY5Y cells.The incorporation of CB [7] further augmented the synergistic effect between chemotherapy and phototherapy.

The In Vivo Assessment of the Antitumor Efficacy
Encouraged by the results of in vitro cytotoxicity determinations, we further investigated the in vivo antitumor effect of synergistic chemotherapy combined with photodynamic therapy using SH-SY5Y cell xenograft models.Firstly, we performed in vivo tumor imaging and evaluated the accumulation behavior of DOX/TPOR 4 @CB [7] 4 on an IVIS Spectrum system by utilizing the intrinsic fluorescence of TPOR.As depicted in Figure 4A,B, after 12 h of intravenous injection, fluorescence signals were observed in three groups of tumor tissues.Notably, a distinct fluorescence signal was captured after a 12 h intravenous injection of DOX/TPOR 4 @CB [7] 4 , which exhibited higher intensity after 12 h compared to both TPOR and DOX/TPOR 4 groups, approximately twice as much.We observed that the fluorescence in the tumor tissue exhibited a gradual decrease subsequent to drug injection at 36 and 48 h (Figure S5).The peak fluorescence intensity persisted even at 24 h.Tumor tissue and major organs were harvested upon sacrificing mice at 24 h post-injection.Examination of the fluorescence images from these harvested materials revealed that DOX/TPOR 4 @CB [7] 4 specifically emitted brighter signals within tumor tissue without any accumulation in healthy tissues (Figure 4C).This finding provides crucial evidence for visualizing the diagnosis and treatment of tumors.Loading CB [7] can effectively enhance tumor-targeting capability while reducing nonspecific capture by healthy organs and minimizing side effects.
therapy in vitro.The combination drug exhibits significant potential in reducing th age of chemotherapy, thereby effectively mitigating systemic toxicity and treatm lated side effects, offering promising advantages for neuroblastoma treatment.The gistic application of photodynamic therapy and chemotherapy can mutually enhan other's efficacy, surpassing the photodynamic effect observed in either TPOR group or DOX/TPOR4 group alone.

The Analysis of Flow Cytometry
To further assess the anti-tumor efficacy of the drug, we employed the ANNE FITC/PI double labeling method to quantify the ratio of apoptosis to necrosis fol PDT treatment [53].As depicted in Figure 3C, in the absence of laser irradiation, nificant differences were observed in apoptosis compared to the control group.Ho 4 h after 2 min of irradiation, both the DOX/TPOR4 group and DOX/TPOR4@CB[7]4 exhibited significant rates of apoptosis at 20.93% and 28.0%, respectively.It is notew that flow cytometry analysis of DOX/TPOR4@CB [7]4 yielded consistent results w MTT assay, demonstrating pronounced cytotoxic effects on SH-SY5Y cells.The in ration of CB [7] further augmented the synergistic effect between chemotherapy an totherapy.

The In Vivo Assessment of the Antitumor Efficacy
Encouraged by the results of in vitro cytotoxicity determinations, we further i gated the in vivo antitumor effect of synergistic chemotherapy combined with ph Furthermore, the efficacy of in vivo Chemo/PDT combination therapy was assessed by monitoring changes in tumor volumes and final tumor weight 18 days after different treatments (Figure 5A).After treatment, the DOX/TPOR 4 group demonstrated superior anti-cancer efficiency compared to the DOX and TPOR groups.The DOX/TPOR 4 @CB [7] 4 group exhibited a significantly greater reduction in tumor volume (Figure 5B), along with enhanced tumor inhibition (84.17%) compared to TPOR (36.69%) and DOX/TPOR 4 (65.95%)groups (p < 0.001) (Figure 5E,F).Throughout the 18-day period, all groups maintained stable body weights without any observed skin damage on their surface skin during treatment on mice surface skin (Figure 5D).
Moreover, tumor slices were subjected to Hematoxylin and Eosin (H&E) staining assay.As depicted in Figure 5G, following an 18-day treatment period, the tumor tissue appeared normal in both the control and CB [7] groups.However, slight damage to the tumor tissue structure was observed in the DOX and TPOR groups.In contrast, evident cell damage accompanied by destruction of the tumor tissue structure was noted in the DOX/TPOR 4 group.Notably, neovascularization and areas of tumor tissue necrosis significantly increased in the DOX/TPOR 4 @CB [7] 4 group.Furthermore, TUNEL staining results demonstrated that apoptosis levels were highest in this particular group compared to others (Figure 6F).Subsequently, serum and urine samples from mice were analyzed for several crucial liver and kidney function indicators after treatment (Figure 6A-E), revealing no significant changes when compared with those of the control group for each indicator tested.Additionally, examination of tissue sections from major organs, including the heart, liver, spleen, lung, and kidney, indicated minimal damage within the treatment group as opposed to that observed within the control group further suggesting absence of any apparent biosafety concerns associated with this drug administration among mice.
compared to both TPOR and DOX/TPOR4 groups, approximately twice as much.W served that the fluorescence in the tumor tissue exhibited a gradual decrease subse to drug injection at 36 and 48 h (Figure S5).The peak fluorescence intensity persisted at 24 h.Tumor tissue and major organs were harvested upon sacrificing mice at 24 h injection.Examination of the fluorescence images from these harvested materials rev that DOX/TPOR4@CB [7]4 specifically emitted brighter signals within tumor tissue wi any accumulation in healthy tissues (Figure 4C).This finding provides crucial evid for visualizing the diagnosis and treatment of tumors.Loading CB [7] can effective hance tumor-targeting capability while reducing nonspecific capture by healthy o and minimizing side effects.Furthermore, the efficacy of in vivo Chemo/PDT combination therapy was ass by monitoring changes in tumor volumes and final tumor weight 18 days after diff treatments (Figure 5A).After treatment, the DOX/TPOR4 group demonstrated sup anti-cancer efficiency compared to the DOX and TPOR groups.The DOX/TPOR4@C group exhibited a significantly greater reduction in tumor volume (Figure 5B), along enhanced tumor inhibition (84.17%) compared to TPOR (36.69%) and DOX/T (65.95%) groups (p < 0.001) (Figure 5E,F).Throughout the 18-day period, all groups tained stable body weights without any observed skin damage on their surface skin ing treatment on mice surface skin (Figure 5D).
Moreover, tumor slices were subjected to Hematoxylin and Eosin (H&E) stainin say.As depicted in Figure 5G, following an 18-day treatment period, the tumor appeared normal in both the control and CB [7] groups.However, slight damage t tumor tissue structure was observed in the DOX and TPOR groups.In contrast, ev The in vivo results have demonstrated that DOX/TPOR 4 @CB [7] 4 exhibits superior tumor accumulation and prolonged fluorescence retention time, thereby enhancing the PDT and chemotherapy for an enhanced anti-tumor effect.This suggests that loading CB [7] improves the tumor-targeting ability and prolongs drug retention in the body, further enhancing the efficacy of TPOR and DOX combination therapy.Additionally, it can serve as a drug carrier to effectively reduce non-specific capture by healthy tissues while passively targeting tumors through enhanced permeability and retention effects, thus reducing toxic side effects.These findings suggest that DOX/TPOR 4 @CB [7] 4 has no significant in vivo organ toxicity with potential as a potent therapeutic agent against cancer with minimal adverse effects.The in vivo results have demonstrated that DOX/TPOR4@CB [7]4 exhibits superior tumor accumulation and prolonged fluorescence retention time, thereby enhancing the PDT and chemotherapy for an enhanced anti-tumor effect.This suggests that loading CB [7]  a drug carrier to effectively reduce non-specific capture by healthy tissues while passive targeting tumors through enhanced permeability and retention effects, thus reducin toxic side effects.These findings suggest that DOX/TPOR4@CB [7]4 has no significant vivo organ toxicity with potential as a potent therapeutic agent against cancer with min mal adverse effects.

Figure 3 .
Figure 3. Killing effect of drugs on SH-SY5Y cells.(A) The cell survival rate of DOX, DOX/TPOR4 and DOX/TPOR4@CB[7]4 groups without laser irradiation and (B) upon irradiat nm, 95.5 mW/cm 2 ) in SH-SY5Y cells measured by MTT (data are presented as mean ± SE (n = Apoptosis rate of SH-SY5Y cells measured by flow cytometry.(D) The statistical analysis g apoptosis rate.

Figure 4 .
Figure 4. Images of drug biodistribution in mice.(A) Fluorescence images of SH-SY5Y tumor ing mice post intravenous injection of drugs for different times.(B) Quantification of fluore intensity of A. (C) Ex-vivo fluorescence images of isolated organs and tumor issue at 24 h injection of drugs.(D) Quantification of fluorescence intensity of C.

Figure 4 .
Figure 4. Images of drug biodistribution in mice.(A) Fluorescence images of SH-SY5Y tumor-bearing mice post intravenous injection of drugs for different times.(B) Quantification of fluorescence intensity of A. (C) Ex-vivo fluorescence images of isolated organs and tumor issue at 24 h postinjection of drugs.(D) Quantification of fluorescence intensity of C.

Figure 5 .
Figure 5. Inhibitory effect of drugs on tumor growth in tumor-bearing mice.(A) Flow chart of photodynamic therapy in mice; (B) tumor growth curves; (C) body weight curves; (D) photographs of tumor-bearing mice at day 0, 7 and 15 treated with different drugs (the red circle indicates the location of the tumor); (E) photographs of the tumor in vitro at the end of treatment; (F) tumor inhibition rate; (G) H&E staining and TUNEL staining of tumors after 18 days of treatments (The red arrow shows the positive result of TUNEL staining, ** p < 0.01，*** p < 0.001).

Figure 5 .
Figure 5. Inhibitory effect of drugs on tumor growth in tumor-bearing mice.(A) Flow chart of photodynamic therapy in mice; (B) tumor growth curves; (C) body weight curves; (D) photographs of tumor-bearing mice at day 0, 7 and 15 treated with different drugs (the red circle indicates the location of the tumor); (E) photographs of the tumor in vitro at the end of treatment; (F) tumor inhibition rate; (G) H&E staining and TUNEL staining of tumors after 18 days of treatments (The red arrow shows the positive result of TUNEL staining, ** p < 0.01, *** p < 0.001).

Figure 6 .
Figure 6.Biosafety analysis of different treatments.Content analysis of Serum renal function in cators Scr (B), BUN (C), urine Ucr (A), and PRO (D).(F) Content analysis of serum liver functi indexes ALT, AST (E).Pathological analysis of major organs in different treatment groups after days (F).Pathological analysis of major organs in different treatment groups after 18 days (F).( n > 0.05)

Figure 6 .
Figure 6.Biosafety analysis of different treatments.Content analysis of Serum renal function indicators Scr (B), BUN (C), urine Ucr (A), and PRO (D).(F) Content analysis of serum liver function indexes ALT, AST (E).Pathological analysis of major organs in different treatment groups after 18 days (F).Pathological analysis of major organs in different treatment groups after 18 days (F).( ns p > 0.05).