All of the chemicals and ingredients were purchased from Sigma-Aldrich (St. Louis, United States) and used without further purification. Cell culture Dulbecco’s modified Eagle’s medium (DMEM), antibiotic, trypsin, phosphate buffer saline (PBS), and fetal bovine serum (FBS) were purchased from HyClone (Texas, United States). Deionized (DI) water (R = 18.0 M Ω) from the Millipore deionizer was used in the entire experimental study. MDA-MB-231 cells and BAMB/c nude mice (6 weeks, 18–20 g, female) were purchased from Korean Cell Line Bank (Seoul, Republic of Korea) and Hana Biotech (Gyeonggi-do, Republic of Korea), respectively.

25 mg/ml of Mn-HAp/FA-IR-783 was combined with distilled water, dispersed using ultrasonic, and exposed to an 808 nm NIR laser for irradiation. The solution’s temperature rise was monitored every 30 s using an infrared laser and infrared thermal imager. In this experiment, the distilled water group was compared with the Mn-HAp/FA-IR-783 group. The stability of Mn-HAp/FA-IR-783 under laser irradiation was further assessed by irradiating 3 ml of the material (200 μg/ml) for five cycles, with each temperature rise lasting 7 min. Each cycle ended with the sample being cooled to room temperature before the next one started. In the experiment, the temperature variations for each sample during each cycle were noted.

To assess the cytotoxicity of in vitro cells, the MTT test was performed. In the experiment, DMEM is carefully mixed with 10% FBS and 1% antibody; MDA-MB-231 cells are counted and diluted before being placed on 96-well plates (seeded cell density in each well 1 × 10⁴ cells). The incubator’s temperature was set to 37°C with 95% air and 5% CO₂. After overnight incubation, the media was mixed with HAp, IR-783 and Mn-HAp/FA-IR-783 (50–250 μg/ml). After a 24-h incubation period, MTT was evenly added to the cells in each well, and they were then treated for 4 h in the dark. To determine the percentage of cell viability, a microplate reader (490 nm) was used to record optical absorbance (OD value). Finally, cell activity was calculated using the following formula: C e l l   v i a b i l i t y   % = O D s a m p l e   / O D c o n t r o l × 100 % Where, O D s a m p l e represents the optical absorbance of cells that had undergone various treatments; and O D c o n t r o l represents the optical absorbance of untreated cells.

The feasibility of ingesting Mn-HAp/FA-IR-783 in cancer cells was studied by CLSM. MDA-MB-231 cells were inoculated into a confocal plate of 1 × 10⁴ cells/well at 37°C for 24 h under the same environment as before, and then the culture medium was changed. After the cells were cultured for 6 h, the media was aspirated. PBS was used to wash cells 3 times. After washing AO/PI Live/Dead cell staining was performed under confocal laser scanning microscopy.

Methylene Blue (MB) was selected as the target material for the detection of extracellular ROS. Mn-HAp/FA-IR-783 was then added to the 5 mM MB solution. After irradiated with 0.75 W cm⁻² 808 nm laser every 5 min interval UV absorption of MB was recorded. To study in vitro ROS effect, MDA-MB-231 cell lines were seeded in 12 well plates. After culturing for 24 h, the culture media was mixed with HAp, Mn-HAp/FA-IR-783 nanoparticles. Post laser treatment (4 h later), diluted 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution was added. The culture plates were incubated 30 min in CO₂ incubator. Finally, the cells were washed four times with serum-free culture medium to remove excess DCFH-DA solution, and observed under confocal microscope.

The in vivo treatment experiment was initiated when the tumor size in the MDA-MB-231-induced nude mice reached 100–120 mm³. The experimental mouse was divided into three groups: Mn-HAp/FA-IR-783 nanoparticles only, Laser only, and Mn-HAp/FA-IR-783 combined with Laser. The nanoparticles were delivered into the mouse’s tail vein using sterile syringes. The dosage was 1.5 mg/kg and was injected into the tail vein of the mouse based on weight. Post 6 h, time period after injection the tumor was irradiated using 808 nm laser. The corresponding PTT images were captured using the infrared thermal camera.

All animal testing was done in compliance with institutional rules and regulations for animal care services that were approved by Pukyong National University in Busan, Republic of Korea (PKNUIACUC-2021-25). The female BALB/c naked mice (18.7 ± 0.7 g), aged between six and 7 weeks were procured from Orient Bio Inc. (Seongnam, Republic of Korea). MDA-MB-231 cells (10⁶ cells) suspended in 100 μl of PBS were injected subcutaneously into the flanks of all the mice. The animals were kept under observation with regular care until the tumors reached a volume of ∼100 mm³, measured using digital calipers. Five groups of animals were separated. For this experiment, a total of 20 animals were used, as shown in Supplementary Table S1.

The animals with tumors received 100 μl of a 200 μgmL⁻¹ Mn-HAp/FA-IR-783 nanoparticle solution through injection. After that, until the active nanoparticles reached the desired tumor site, the mice were kept in a suitable environment and under regular observation. After each 2 h interval the animals were studied under the fluorescence imaging system to identify the tumor region. After 6 h interval sufficient fluorescence signals received from the tumor region. An optical fiber connected to an 808 nm laser diode was used to deliver 7 min of 0.75 W cm⁻² radiation to each mouse’s tumor. All five groups, which include (I) control group without tumor, (II) tumor bearing mouse without any treatment (III) tumor bearing mouse with laser irradiation only (IV) Mn-HAp/FA-IR-783 nanoparticles injected mouse, and (V) Mn-HAp/FA-IR-783 injected mice with laser irradiation. Before irradiation, a probe thermometer was placed into the center of the tumor. The temperature of the tumor was recorded during the irradiation at 1 s intervals.

Following the completion of the 3-week treatment, the mice from each group were euthanized, and the tumor as well as the five major organs (kidney, liver, spleen, heart, and lungs) were harvested. The experimental organs were washed in saline water and stored in 10% formalin for fixing and staining. The tissues were immersed for 24 h, preserved with paraffin, and cut into 4 m-thick slices for hematoxylin and eosin (H&E) staining. The histology slides were then seen under a microscope.

Each result was presented as a mean standard deviation (SD). Using the program Origin 9.0, all of the charts in this article were produced. Analysis of variance was used to statistically determine whether the experiment’s results were similar (Student t-test). The p-value < 0.05 cutoff was used to determine statistical significance.

The XRD analysis of HAp and Mn-HAp (Mn: 0.5, 1.0, 2.0 mol%) nanoparticles were studied and compared with the standard JCPDS (# 00-009-0432) data of the pristine HAp (Figure 2A). Sharp peaks on XRD patterns show that the calcined HAp nanoparticle has good crystallinity. The maximum intensity peak was found at 2θ value of 31.7°, corresponding to (211) planes for pristine HAp. The diffraction peaks for HAp at 25.5°, 28.6°, 32.3°, 39.2°, 46.4°, 49.1°, 53.4°, 61.3°, and 64.1°, respectively, indicate the computed (002), (210), (112), (130), (222), (213), (004), (214), and (304) planes (2 diffraction angle). Debye Scherer’s relation was used to determine the average crystallite size of the HAp particles over the peak with the highest intensity (211): D = 0.9 λ   / β ⁡ cos ⁡ θ where D stands for average crystallite size, FWHM (full width at half maximum) of the peak, diffraction wavelength (0.154059 nm), and diffraction angle are all mentioned. Table 1 shows the typical crystallite size of calcined HAp and various Mn-HAp (Mn: 0.5, 1.0, 2.0 mol%).

The synthesized Mn-HAp nanoparticles were subjected to diffuse reflectance spectroscopy, in which the valence electrons are excited to empty orbitals using visible light. Figure 2B shows the UV-vis DRS absorption spectra of produced HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%). There are no significant absorption peaks found for HAp material alone. Whereas a distinct broad absorption band was found for materials containing varying amounts of Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) (in the range of approximately 380–430 nm). This investigation supports the successful doping of manganese on the HAp matrix.

TG-DT analysis was used to determine the thermal stability of HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%) using an alumina crucible heated to 800°C at a heating rate of 10°C/min (Figure 2E). Pure HAp has a mass loss of about ∼22.65%. Contrarily, mass losses of ∼21.75%, ∼19.89%, and ∼12.73% were found for the other samples Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%), which are comparably lower than HAp. The doping of Mn constantly increases the thermal stability.

Additionally, the differential thermal analysis curve for HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%) are represented in Figure 2E. The differential thermal curves of each Mn-HAp sample have a similar shape at temperatures between approximately 55 and 515°C, and the HAp graph exhibits a similar sort of curve at temperatures between 125 and 425°C. There were, however, no discernible exothermic or endothermic processes. The TG-DTA graph supports the Mn-HAp nanoparticles’ heat stability.

The ultrastructure and their corresponding TEM and EDS analysis of the synthesized HAp and Mnx–HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles is represented in Figures 2F–O. The synthesized nanoparticles show elongated rod-shaped structure with average length of ∼26 ± 2.8 nm for pristine HAp (Figure 2F), ∼26 ± 3.2 nm for 0.5 mol% Mn-HAp (Figure 2G), ∼27 ± 3.1 nm for 1.0 mol% Mn-HAp(Figure 2H, ∼28 ± 2.1 nm for 2.0 mol% Mn-HAp (Figure 2I) nanoparticles. The HR TEM study revealed an atomic distance of 3.4 Å (002), 2.8 Å (211), 3.4 Å (002), 2.8 Å (211) which corresponds to the hydroxyapatite plane for HAp and Mnx–HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles respectively (Figures 2K–N). Figure 2N, shows the atomic distance of 2.4 Å (121) associated to the Mn plane. The elemental analysis confirmed the presence of “Ca” and “P” due to HAp and “Mn” due to doping materials (Figures 2J and O). The detailed EDS analysis is represented in supplementary section (Please see the supplementary data Supplementary Figure S1).

The synthesized HAp and Mnx-HAp (x = 0.5, 1.0, and 2.0 mol%) nanoparticles, shown in Figure 2D, were examined using FTIR spectroscopy. The nanoparticles exhibit a variety of peaks in their FTIR spectra, which are described as follows. In all samples, the bands at 966–1,099 cm⁻¹ and 573–601 cm⁻¹ are related to the P-O bond caused by phosphate groups (Vinoth Kumar et al., 2021). The phosphate group’s v3 stretching mode is responsible for the peak at 1,041 cm⁻¹. Peak P-O tension ν1 stretching took place at 966 cm⁻¹ (Park et al., 2022). The v4 bending vibration based on the phosphate group is shown in the peak at 573 cm⁻¹. The band found between 3,570 cm⁻¹ and 638 cm⁻¹ demonstrated the hydroxyl group (O-H) correlating to the O-H stretching of HAp (Prasanna and Venkatasubbu, 2018). Additionally, the peaks for the phosphate group and characteristic vibration in produced nanoparticles are unaltered.

In order to investigate the HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%), detailed chemical structure, phase, and molecular interactions are studied using the effective non-destructive method of Raman spectroscopy. HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%) functional groups, bands, and modes’ Raman spectra are shown in Figure 2C. Both samples displayed similar functional groups and modes to the HAp and Mnx-HAp nanoparticles (x = 0.5, 1.0, and 2.0 mol%). The ʋ1(PO₄) band at 962 cm⁻¹ is very intense which is exactly similar with HAp and all the different concentration Mn doped HAp nanoparticles. The Raman active modes were detected at 1,033 cm⁻¹, 1,046 cm⁻¹, 1,076 cm⁻¹ (ʋ3(PO₄)), 502 cm⁻¹, 581 cm⁻¹, 592 cm⁻¹ (ʋ4(PO₄)), and 432 cm⁻¹, 447 cm⁻¹ (ʋ2(PO₄)), respectively (Yilmaz and Evis, 2014). However, when Mn was doped on HAp nanostructures, peak locations were changed. Additionally, there is a strong correlation between the FTIR data and the peak positions determined by the functional groups.

PDT can cause tumor cells to die by overproducing reactive oxygen species (ROS), which are created when photosensitizer (PS), light, and oxygen combine. Mn²⁺ may be transformed into Mn(OH)₂ in the presence of H₂O₂ in a neutral environment, and the unstable Mn(OH)₂ could subsequently be oxidized by H₂O₂ to produce MnO₂, according to previous Mn²⁺ studies (Fu et al., 2019; Liang et al., 2022). The catalytic breakdown of H₂O₂ to produce oxygen and simultaneously reduce to Mn²⁺ can be strongly induced by the created MnO₂ (Figure 3C). The following reactions show the entire process: M n 2 + + 2   H 2 O 2   ⇄   M n ( O H ) 2 + 2 H + M n ( O H ) 2   + H 2 O 2   →   M n O 2   + 2 H 2 O M n O 2 + H 2 O 2 + 2 H +   →   M n 2 + + 2 H 2 O + O 2   ↑

In this present study HAp and Mnx-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) nanoparticles were synthesized rod shaped with a high surface area (Please see the supplementary section for BET surface area analysis, Supplementary Figure S2). The synthesized nanoparticles further studied for PDT efficiency study (Figures 3A,b1,b2). Here, Methylene blue dye was chosen as targeted dye. After inoculation of different concentration nanoparticles, the solution was irradiated under different light wavelengths. After optimization 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles were used for in vitro and in vivo study. In vitro ROS activity was monitored with MDA-MB-231 cell lines incubated with 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles. The culture media was combined with HAp, Mn-HAp/FA-IR-783 nanoparticles after being cultured for 24 h. Post laser treatment (4 h later), diluted 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution was added. The amount of solution used completely covers the acquired cells. After maintaining the same condition, the cells were incubated for 30 min. In order to remove excess DCFH-DA solution, the cells were washed three times with serum-free growth media before being examined under a confocal laser scanning microscope (Figure 3D). After treated with laser the cells emit green fluorescence due to the generation of singlet oxygen ¹O₂. This experimental study confirms the PDT efficiency of synthesized 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles.

After 24 h of incubation with various concentrations, the MTT assay of HAp and Mnx-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) nanoparticles exhibited non-toxic properties to the MDA-MB-231 cell line (Figure 4A). More than 90% of the cells remained alive at the lowest dose (50 μg∙mL⁻¹), whereas a greater concentration of 250 μg∙mL⁻¹ resulted in somewhat higher toxicity which results ∼17% dead cells. As the highest concentration (250 μg mL⁻¹) likewise produced ∼82% cell viability, results HAp and Mnx-HAp/FA-IR-783 nanoparticles (x = 0.5, 1.0, and 2.0 mol%) exhibit very minimal toxic behavior up to this concentration. A similar investigation was carried out using 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles, incubated with 200 μg∙mL⁻¹ concentrations for 24 h, 48 h, and 72 h with the MDA-MB-231 cell line. When the Mn-HAp/FA-IR-783 concentration reaches 250 μg∙mL⁻¹, little toxicity was observed (overall cell viability is ∼85%). Finally, the selected concentration of 200 μg∙mL⁻¹ showed ∼86% cell viability (Figures 4A,C). The MTT study further extended with 200 μg mL⁻¹ dose concentration of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles irradiated with variable laser power (0.5 W cm⁻², 0.75 W cm⁻², and 1.0 W cm⁻²) (Figure 4C). The control group (without any nanoparticles) shows the highest cell viability, whereas only HAp also shows more than ∼90% viability while irradiated with different laser power densities. The MTT results confirmed all the Mn_x-HAp/FA-IR-783 (x = 0.5, 1.0, and 2.0 mol%) are lethal while irradiated even with minimum power laser (0.5 W∙cm⁻²). Approximately 95% cells were dead when 0.75 W∙cm⁻² laser power were used. With increasing laser power intensity, the materials become more lethal. In addition, control group and post PTT/PDT treated MDA-MB-231 cell line groups were assessed by acridine orange/propidium iodide (AO/PI) staining under confocal laser scanning microscopy (Figure 4D, E). To confirm the effect of different laser power density Flow cytometry study was performed.

The flow cytometry was carried out with HAp and 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles treated MDA-MB-231 cell line irradiated with 0.75 W cm⁻² laser (Figure 4B). The only HAp nanoparticles and laser exposure present in the study of control cells. For the control group, the flow cytometry investigation showed ∼87.5% cells in healthy condition followed by ∼3.16% in the early and ∼6.64% in late apoptosis stage (very less amount ∼2.70% are in necrosis stage due to may be normal cell death). For 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles irradiated with 0.5 W cm⁻² laser (for 7 min) treated cells shows ∼95.2% cells in late apoptosis condition with ∼1.95% in early apoptosis phase. Very little amount (∼0.1%) was observed in necrosis stage, whereas ∼2.76% cells still shows healthy behavior. With increasing laser power intensity of 0.75 W cm⁻² the treated cells show ∼95.6% in late apoptosis, ∼1.81% in early apoptosis, and remaining very few cells (∼2.56%) shows viability. While using 1.0 W cm⁻² laser-irradiation, only ∼0.91% cells were found to be healthy with ∼95.7% in late apoptosis phase, and remaining ∼3.41% in necrosis stage. The FACS study is well corelated with MTT assay which confirms the nanoparticles reproducible performance (Figures 4B,C).

Heparin-stabilized blood samples collected from BALB/c nude mice and further extracted by centrifugation were used for the hemocompatibility assay. Red blood cells (RBCs) were collected and diluted with 10X PBS buffer. Various quantities of 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles were added to the RBC suspension (500 μl) at 50, 100, 150, 200, and 250 μg/ml, with medical saline solution served as the negative control and distilled water as the positive control, respectively. All of the solutions were vortexed, allowed to incubated for 4 h at 37°C, and then centrifuged for 5 min at 5,000 rpm. Each sample’s absorbance was measured at a wavelength of 541 nm using a Tecan Infinite F50 microplate reader. The following equation was used to calculate the hemolysis of RBCs in each sample: H e m o l y s i s   ( % ) = A b s S a m p l e − A b s N e g a t i v e   c o n t r o l A b s P o s i t i v e   c o n t r o l − A b s N e g a t i v e   c o n t r o l  

2.0 mol% Mn-HAp/FA-IR-783 nanoparticles were injected intra venous tail vein region for in vivo cancer therapy. Therefore, it is crucial to look into whether the injected nanoparticle is compatible with blood or not. As shown in Figure 4F, 2.0 mol% Mn-HAp/FA-IR-783 demonstrated outstanding blood compatibility with very less hemolytic activities of ∼5.67 ± 0.14% even at a high concentration of 250 μg mL⁻¹.

The florescence imaging study was performed by indigenously developed LUX 4.0 imaging system (LUX 4.0; OHLABS, Busan, Republic of Korea). In vitro fluorescence imaging study was performed with 2.0 mol% Mn-HAp/FA-IR-783 nanoparticles incubated with MDA-MB-231 cell lines. First, MDA-MB-231 cell lines were left incubated for 24 h for culture plate attachment. Next different concentration (25, 50, 75, 100 μg/ml) prepared nanoparticles were inoculated to the nanoparticles for 4 h. Post incubation the 96 well plates were imaged under LUX 4.0 imaging system (Figure 5B). The study extended with in vivo BALB/c mouse model. The tail vein was used to administer 100 μl of 2.0 mol% Mn-HAp/FA-IR-783 (100 μg/ml) to the tumor-bearing nude mice, thereafter fluorescence monitoring (each 2 h interval up to first 12 h, followed by each 24 h interval) was performed using LUX 4.0 imaging system (Figure 5A). The nanoparticle shows excellent fluorescence property under imaging system. The interesting in vivo study revealed the folic acid conjugated nanoparticles excellent targeting specifically to the tumor region. In this study Group IV animals (only nanoparticles injection) were selected for the imaging purposes. The injected 2.0 mol% Mn-HAp/FA-IR-783 have excellent targeted imaging capabilities could be a promising feature for clinical level imaging guided cancer therapy.

The PTT experiment was started by exposing the tumors to a 0.75 W cm⁻² laser (808 nm) irradiation for 7 min. The performance of the nanoparticle is shown in Figure 6A. PTT stability of Mn-HAp/FA-IR-783 under repeated five cycles of 808 nm laser (0.75 W.cm-2; ON/OFF) (Figure 6B). The test mice were split into five groups, of which groups III and V received laser therapy. Furthermore, no nanoparticles were administered via injection to the mice in groups I, II, or III. Since no heat generation occurred in the absence of nanoparticles, it was confirmed by the in vitro study that PTT was ineffective for groups I, II, and III. No laser irradiation was used on groups I or II. The initial experimental investigation began at 25 ± 2°C, although the mouse’s body temperature was 30.6 ± 1.2°C at the beginning. The animals in group II demonstrated a maximum temperature increase of 1.6°C after being exposed to a 0.75 W cm⁻² laser (808 nm) for 7 min, resulting in a final body temperature of 32.2 ± 0.7°C. The laser dosage alone was unable to raise the temperature because there were no nanoparticles present inside the target tumor location (Mondal et al., 2022). Intratumorally injected with 100 μl of a 200 μg/ml 2.0 mol% Mn-HAp/FA-IR-783 solution, Group V animals were then subjected for 7 min to a 0.75 W cm⁻² laser (808 nm). According to Figure 6C, IR thermal images, the photothermal treatment causes the mouse’s body temperature to rise from 30.6 to 55.2°C. After the photothermal treatment, the tumor area once more recorded a temperature of 31.4°C, which is close to the mouse’s normal body temperature. The calculated photothermal conversion efficiency of 2.0 mol% Mn-HAp/FA-IR-783 to be η ≈ 31.2% which is effective for sufficient heat generation (Please see supplementary information, Supplementary Figure S3). The tumor region’s temperature increase was more than enough to effectively ablate the tumors in vivo. This therapy’s safety is confirmed by the fact that no animals died after receiving treatment. Aside from that, the photothermal shock had no discernible effects on the mice’s behavior or body weight. Our investigation found no discernible toxicity or deadly effect, supporting earlier reports’ findings (Terentyuk et al., 2010). All of the mice were monitored for a full 3 weeks following treatment to ensure that the photothermal effects had fully healed (Figure 6D).

From tumor induction to photothermal treatment, the in vivo experimental study was carried out with a total time period of 7 weeks (Figure 7A). After PTT therapy, all of the mice were monitored and cared for 3 weeks, until full recovery. After the study post-PTT surveillance was completed, the histological examination was carried out. The primary organs were removed and prepared for Hematoxylin and eosin staining as described in the experimental part, including the kidney, liver, heart, spleen, and lungs (Figures 7A,B). After that, an optical microscope was used to look for any physiological and structural alterations in the organs and the tumor. The morphologies of all the cross-sectional tissues appeared to be in good health, and no such abnormalities were seen in any of the organs. Additionally, no discernible color change was seen, therefore all experimental groups’ organs looked to be normal (I-V). Figure 7B displays the tissues’ histological images. The general harmless character of 2.0 mol% Mn-HAp/FA-IR-783 therapies is well supported by the absence of tissue damage, aberrant behavior, and body weight changes. While examining the tumor tissues’ histological section, we noticed several substantial PTT-related alterations. In contrast to the untreated tumors (groups II and IV), which have poorly differentiated cells with granulated deep stained nuclei, the tumors in the 2.0 mol% Mn-HAp/FA-IR-783 treated animals (group III) display medium sized tumors with deep stained cells. Finally, animals treated with 2.0 mol% Mn-HAp/FA-IR-783 and laser irradiation (group V) are completely healed without any tumor recurrence. There were no malignant tissues found in the treated animals of group V when the post-treatment surrounding tissues were collected, only healthy cells with distinct nuclei were observed. All tumor volumes and animal body mass were measured throughout the monitoring period at regular intervals (Figures 7D,E).

At 24-h and 3-week intervals, mice from treatment groups were euthanized in order to study the accumulation of Mn-HAp/FA-IR-783 in organs. The organs underwent an 8-h HNO₃ treatment after being dissected. Inductively coupled plasma mass spectrometry was utilized to measure the Mn concentration in each group (ICP-MS). The findings demonstrate a significant level of Mn, post 24 h tumor treatment (Figure 7C). At 24 h, the estimated cumulative post-injection dose per gram of tumor tissue was around 1.2% ID∙g⁻¹. Due to intravenous injection, the body’s various organs were accumulating the nanoparticles after they mixed with blood flow. The spleen therefore accumulated the most (∼0.28% ID∙g⁻¹ at 24 h and ∼0.07% ID∙g⁻¹ after 3 weeks), while the kidney, liver, lungs, and heart accumulated, respectively 0.21, 0.21, 0.10, and 0.08% ID∙g⁻¹ 24 h post treatment. After 3 weeks, a biodistribution analysis showed that the nanoparticles had been significantly cleared from the body systems, with extremely minute levels found in the spleen, kidney, liver, lungs, and heart, respectively: 0.04, 0.02, 0.05, 0.01, and 0.02% ID∙g⁻¹. There were no hazardous or fatal effects as a result of the extremely low nanoparticle concentration inside the organs. The study as a whole concludes that the doses of nanoparticles used here are harmless, nonlethal, and extremely effective for PTT/PDT cancer therapy.