Black phosphorus quantum dots camouflaged with platelet-osteosarcoma hybrid membrane and doxorubicin for combined therapy of osteosarcoma | Journal of Nanobiotechnology

Examination of OPM properties on BPQDs-DOX@OPM

The cellular hybrid membrane-encapsulated nanoparticles consist of two main parts: nanocarrier core and surface cell membrane shell structure [32]. Because the hybrid membrane can retain glycoproteins, lipids, and proteins on the entire cell membrane surface, it can endow the nanoparticles with functions and properties inherent to the source cell. For example, CD47 on the platelet surface can interact with immune cells to inhibit immune clearance [27], and cancer cells can be homotypically targeted [28]. To examine the proteins on BPQDs-DOX@OPM, we examined the hybrid membrane whole protein and signature protein expression by SDS-PAGE gel electrophoresis and Western blot, respectively. The SDS-PAGE gel electrophoresis results are shown in Fig. 2d; they indicate that the BPQDs-DOX@OPM group has both OCM and PM on the protein. The Western blot results are shown in Fig. 2e; they indicate that the OCM, PM, and BPQDs-DOX@OPM groups showed the expression of the signature protein EGFR, signature protein CD47, and both EGFR and CD47, respectively. The results of SDS- PAGE gel electrophoresis and Western blot indicated that BPQDs-DOX@OPM contained an OPM heterodimeric membrane with full protein expression.

Fig. 2
figure 2

BPQDs-DOX@OPM characterization. a Transmission electron micrographs of BPQDs, OPM, BPQDs@OPM, and BPQDs-DOX@OPM (size: 50 nm). b, c Particle size and zeta potential of each group (A: BPQDs, B: OPM, C: BPQDs@OPM, D: BPQDs-DOX@OPM). d SDS-PAGE gel electrophoresis result (A: OCM, B: PM, C: BPQDs-DOX@OPM). e Western blot analysis result (A: OCM, B: PM, C: BPQDs-DOX@OPM). f, g Photothermal performance and temperature rise curve of BPQDs-DOX@OPM. h DOX release characteristics of BPQDs-DOX@OPM

BPQDs-DOX@OPM-like characteristics

In cell membrane-mimetic encapsulation of small-sized nanocarriers, the size of the cell membrane-modified nanocarriers is usually close to that of the cell membrane, and the surface potential of the drug delivery system is close to that of the cell membrane [33, 34]. The TEM results (Fig. 2a) showed that the BPQDs as well as BPQDs@OPM hybrid membrane vesicles had a rounded morphology and good dispersion, and OPM had a shell structure that completely encapsulated multiple BPQDs. The TEM results (Fig. 2b) showed that the particle sizes of the BPQDs, OPM, BPQDs@OPM, and BPQDs-DOX@OPM groups were 4.53 ± 1.36 nm, 58.96 ± 13.65 nm, 54.22 ± 17.69 nm, and 68.80 ± 23.78 nm, respectively; the particle size of BPQDs-DOX@OPM was close to that of OPM vesicles. The zeta potentials (Fig. 2c) of BPQDs, OPM, BPQDs@OPM, and BPQDs-DOX@OPM as measured by DLS were − 18.07 ± 0.46 mv, − 9.43 ± 0.78 mv, − 4.64 ± 0.34 mv, and − 7.70 ± 0.46 mv, respectively; the zeta potential of BPQDs-DOX@OPM was close to that of OPM.

Photothermal conversion performance of BPQDs-DOX@OPM

Zero-dimensional BPQDs with a 0–10 nm size exhibit a wide absorption range throughout the visible region. Consequently, they have good photothermal conversion efficiency [35]. However, BPQDs are susceptible to oxidative degradation in an environment containing oxygen and water; biofilm encapsulation can isolate the internal BPQDs from oxygen and water to improve their stability [36]. As shown in Fig. 2f and g, After exposing the BPQDs and BPQDs-DOX@OPM to the air environment for 4 days, each set of solutions was irradiated with near-infrared light (808 nm, 1.0 W/cm2) for 10 min. The temperature of the PBS group did not change significantly. By contrast, temperatures of the BPQDs and BPQDs-DOX@OPM groups increased rapidly and reached 41.7 °C and 52.3 °C, respectively, after 600 s. Further, the final temperature of the BPQDs-DOX@OPM was higher than both the BPQDs and the tissue cell lethal temperature of 48 °C [11].

BPQDs-DOX@OPM drug slow release curve

The calculated drug loading and encapsulation efficiency were 36.2% and 73.4%, respectively, based on the linear regression equation of the DOX standard curve. As shown in Fig. 2h, the 72-h release of DOX from BPQDs-DOX@OPM and BPQDs-DOX was 34.88% and 61.53%, respectively; this indicated that the OPM hybrid film encapsulation system could slow down the release of DOX and help prolong the half-life of BPQDs-DOX@OPM in the blood circulation system. After irradiation with 808-nm NIR light, owing to photostimulation-responsive release, the 72-h DOX release of the BPQDs-DOX@OPM(+) group was 76.04%; this was significantly higher than that of the BPQDs-DOX@OPM group without light irradiation.

In vitro stability assessment of BPQDs-DOX@OPM

Zero-dimensional BPQDs, with sizes ranging from 0 to 10 nm, demonstrate broad absorption across the visible region, indicating favorable photothermal conversion efficiency. However, BPQDs are susceptible to oxidative degradation in the presence of oxygen and water. Biofilm encapsulation technology was employed to isolate the internal BPQDs from oxygen and water to enhance their stability. To evaluate the enhanced stability conferred by OPM encapsulation, BPQDs and BPQDs-DOX@OPM, both with consistent concentrations, were dispersed in water and exposed to air for 8 days. Optical property measurements were conducted at specific intervals (0, 2, 4, 6, and 8 days).

Figure 3a, b illustrates the typical broad absorption bands in the UV and NIR regions exhibited by both BPQDs and BPQDs-DOX@OPM. However, the absorbance intensity of BPQDs in water decreased over time, whereas no significant decrease in absorbance intensity was observed for BPQDs-DOX@OPM. The percentage change in absorbance intensity at 808 nm for BPQDs and BPQDs-DOX@OPM is depicted in Fig. 3c, d, respectively. After 2 days, the absorbance intensity of BPQDs decreased by 23.4% ± 4.47%, and after 8 days, it decreased by 72.3% ± 0.71%. This decrease was attributed to the degradation of bare BPQDs, resulting in diminished absorbance. In contrast, the absorbance of BPQDs-DOX@OPM remained more stable, with only a 6.57% ± 2.14% decrease after 8 days. This enhanced stability can be attributed to the shell structure of OPM, which reduces the influence of oxygen and water on BPQDs, thereby improving the stability of BPQDs-DOX@OPM under environmental conditions.

Fig. 3
figure 3

UV–Vis absorption spectra of BPQDs and BPQDs-DOX@OPM at different time points and the corresponding temperature rise curve. a Absorption spectra of BPQDs at 0, 2, 4, 6, and 8 days; b absorption spectra of BPQDs-DOX@OPM at 0, 2, 4, 6, and 8 days; c statistical analysis of the absorbance of BPQDs at 808 nm at different time points; d statistical analysis of the absorbance of BPQDs-DOX@OPM at 808 nm at different time points; e temperature rise curves of BPQDs on days 0, 2, 4, 6, and 8 days; f temperature rise curves of BPQDs-DOX@OPM on days 0, 2, 4, 6, and 8 days

Figure 3e presents the temperature rise curve of BPQDs, showing a decrease over time due to the unstable nature and easy oxidative degradation of BPQDs. Conversely, Fig. 3f demonstrates that the temperature rise curve of BPQDs-DOX@OPM does not exhibit a significant decrease over time. This observation aligns with the results obtained from the UV–vis absorption spectra of BPQDs-DOX@OPM, confirming that the OPM shell structure enhances the stability of BPQDs under environmental conditions.

In vitro biocompatibility of BPQDs@OPM drug delivery system

Cytotoxicity of BPQDs@OPM drug delivery system on Saos-2 cells

The in vitro biocompatibility assessment of nanocarriers regarding tissue cytotoxicity and hemocompatibility is considered the most reliable and simplest technical tool in biosafety assessment [37]. Studying the dose- and time-dependent cytotoxicity of nanocarriers is one of the most common methods to detect cytotoxicity in vitro [38]. Therefore, as shown in Fig. 4a, Saos-2 was plated for 12 h after BPQDs@OPM treated cells. The results of the CCK-8 cell proliferation activity assay showed that the relative cell survival rate of each group was maintained above 95% after coincubation of BPQDs@OPM with cells at different concentrations for 24 h, and the difference was not statistically significant. As shown in Fig. 4b, after 12 h of Saos-2 cell plating, the BPQDs@OPM system was coincubated with Saos-2 cells for 6, 12, 24, 48, and 72 h, and the CCK-8 cells were assayed for relative cell viability. The assay results showed that the cell proliferation activity of the BPQDs@OPM drug delivery system was maintained above 95% at different periods of coincubation with Saos-2 cells, and the differences were not statistically different. The dose- and time-effect relationships of BPQDs@OPM both indicated its high biocompatibility.

Fig. 4
figure 4

BPQDs-DOX@OPM in vitro performance study. a Cell viability of BPQDs@OPM with different BPQD concentrations after coincubation with Saos-2 for 24 h. b Cell viability of BPQDs@OPM with BPQD concentration of 0.2 mg/mL after coincubation with Saos-2 cells for different periods. c Photographs of supernatants of BPQDs and BPQDs-DOX@OPM hemolysis experiments. d Calculation of hemolysis rates of BPQDs and BPQDs-DOX@OPM. e DID fluorescence-labeled BPQDs-DOX@OPM after coincubation with Saos-2, 3T3, and HBMEC, and RAW264.7 for 4 h. Laser confocal microscopy imaging (size: 50 μm)

Hemocompatibility of BPQDs@OPM drug delivery system

Chemotherapeutic drug carriers are mostly delivered to the tumor site through blood circulation. Investigating their hemocompatibility can help further understand the safety and efficacy of drug delivery systems [39, 40]. Therefore, the erythrocyte hemolysis assay was chosen to verify the BPQDs@OPM hemocompatibility in this study. As shown in Fig. 4c and d, after coincubation of each treatment group with the erythrocyte suspension for 1 h, the erythrocyte hemolysis rates of BPQDs@OPM groups with BPQD concentrations of 0.05, 0.1, 0.2, and 0.4 mg/mL were below 3%. The BPQDs group with 0.1 mg/mL BPQD concentration showed the highest erythrocyte hemolysis rate of 3.10 ± 0.75%. The hemolysis rate of BPQDs and BPQDs-DOX in all concentration groups was less than 5%, in accordance with the required hemolysis rate for biological materials; this indicated that both BPQDs and BPQDs@OPM had good hemocompatibility.

In vitro targeting study of BPQDs-DOX@OPM

As shown in Fig. 4e, surface antigens are reportedly responsible for the homotypic adhesion of cancer cells, and the cancer cell membrane camouflage platform exhibits ideal homotypic cancer-targeting ability [41]. CD47 on the platelet surface can signal “do not eat me” to phagocytes, thus gaining immune evasion ability and conferring a longer half-life to the drug delivery system [42]. To verify the isotype targeting ability and immune evasion capacity of BPQDs-DOX@OPM, the cellular uptake of DiD-labeled BPQDs-DOX@OPM was evaluated using Saos-2, 3T3, HBMEC, and RAW264.7 as controls. DID-(BPQDs-DOX@OPM) exhibited strong red fluorescence after coincubation with Saos-2 cells for 4 h. No significant fluorescence distribution was seen in 3T3, HBMEC, and RAW264.7 cells, indicating that BPQDs-DOX@OPM has OS cell isotype targeting and immune evasion abilities.

In vitro anti-tumor effect study of BPQDs-DOX@OPM

Effect of BPQDs-DOX@OPM on proliferative activity of Saos-2 cells

Necrosis and apoptosis are the most common forms of cell death. To investigate the in vitro anti-tumor effect of the BPQDs-DOX@OPM combined drug delivery system, we detected the proliferative activity of Saos-2 by CCK-8 and the apoptosis rate by flow cytometry. As shown in Fig. 5a, different groups were set (i.e., Control, BPQDs, BPQDs@OPM, DOX, BPQDs-DOX, and BPQDs-DOX@OPM), and each group was irradiated with 808-nm NIR light when adding the drug. After treating Saos-2 cells for 24 h, the cell survival rates of the BPQDs and BPQDs@OPM were 82.32% ± 1.54% and 72.48% ± 1.30%, respectively. The cell survival rate in the BPQDs@OPM group was lower than that in the BPQDs group, indicating that the surface modification of the OPM could enhance the stability of BPQDs. BPQDs-DOX@OPM was significantly lower, indicating that the combined treatment had a more potent killing effect on OS.

Fig. 5
figure 5

BPQDs-DOX@OPM in vitro efficacy study. a Each treatment group treated with Saos-2 cells for 24 h, relative cell survival rate. b Apoptosis rate of Saos-2 cells treated with each treatment group for 24 h. c Statistical analysis of apoptosis rate of Saos-2 cells. (*P < 0.05,**P < 0.01, ****P < 0.001)

Effect of BPQDs-DOX@OPM combined drug delivery system on apoptosis of Saos-2 cells

As shown in Fig. 5b, c, different groups (i.e., Control, BPQDs, BPQDs@OPM, DOX, BPQDs-DOX, and BPQDs-DOX@OPM) were set up to treat Saos-2 cells for 24 h. Each group was irradiated with 808 nm NIR light when the drug was added. Compared with the apoptosis rate of the control group, those of the DOX, BPQDs@OPM, and BPQDs-DOX groups were 28.00 ± 1.38%, 30.36 ± 2.64%, and 36 ± 1.35%, respectively. The BPQDs-DOX@OPM combined treatment group had an apoptosis rate of 42.97 ± 1.13%, indicating that the combined therapy had a more powerful killing effect on OS than single-agent therapy.

In vivo targeting study of BPQDs-DOX@OPM

In vivo targeting assay with in vivo optical imaging of small animals

To further confirm the hypothesis of targeting, the BPQDs-DOX@OPM combination drug delivery system was labeled with the fluorescent dye Cy5, and its in vivo biodistribution was detected by in vivo fluorescence imaging of small animals. Figure 6a, b shows that BPQD-DOX partially accumulated in tumor tissues at 6 and 12 h after drug injection compared with the NS group. This is due to the “passive targeting” effect [43], and the aggregation of BPQDs-DOX in tumor tissues and major organs decreased at 48 h, probably owing to its smaller size and rapid metabolic clearance by the body. Compared with BPQDs-DOX, BPQDs-DOX@OPM showed significant aggregation in tumor tissues 24 h after intravenous injection, and its aggregation in tumor tissues did not show a significant decrease after 48 h. As shown in Fig. 6c, d, tumors and major organs (i.e., heart, liver, spleen, lungs, and kidney) were collected 48 h after drug treatment. The fluorescence imaging results showed that no obvious fluorescence distribution was observed in all major organs of the BPQDs-DOX-treated group, and the fluorescence intensity in the tumor tissues was low, indicating that BPQDs-DOX was rapidly cleared by the organism at 48 h. The BPQDs-DOX@OPM-treated group showed an obvious fluorescence distribution in the tumor tissue but not in the main organs. This indicated that BPQDs-DOX@OPM has good in vivo targeting and long circulation time.

Fig. 6
figure 6

BPQDs-DOX@OPM in vivo targeting study. a, b Fluorescence intensity and distribution in animals after tail vein injection of cy5-labeled BPQDs-DOX@OPM at 6, 12, 24, and 48 h. c, d Fluorescence distribution of tumor tissues and heart, liver, spleen, lungs, and kidneys of animals 48 h after tail vein injection of cy5-labeled BPQDs-DOX@OPM. e Fluorescence distribution of DOX in animal tumor tissues 48 h after tail vein injection of cy5-labeled BPQDs-DOX@OPM. f Analysis of DOX fluorescence intensity in animal tumor tissues

DOX aggregation in tumor tissues

As shown in Fig. 6e and f, 48 h after OS mice were treated with NS, BPQDs-DOX- and BPQDs-DOX@OPM, the tumor tissues were taken to make frozen sections, and the sections were restained with DAPI and observed under a fluorescence microscope. The bright fluorescence of DOX was observed in the BPQDs-DOX@OPM group, and the fluorescence intensity was significantly stronger than that of the BPQDs-DOX group; this confirmed the excellent targeted drug delivery performance of BPQDs-DOX@OPM.

In vivo temperature rise curve of BPQDs-DOX@OPM

To further investigate the in vivo photothermal performance of the BPQDs-DOX@OPM co-loading system, three groups were established: NS saline group, BPQDs group, and BPQDs-DOX@OPM group. Figure 7a, b depicts that each group was subjected to 808 nm near-infrared light irradiation for 10 min. The NS group showed no significant temperature change, while the BPQDs and BPQDs-DOX@OPM groups exhibited a rapid temperature increase during irradiation. After 10 min, the BPQDs and BPQDs-DOX@OPM groups reached 42.7 °C and 48.9 °C, respectively. The final temperature of the BPQDs-DOX@OPM group was higher than that of the BPQDs group, surpassing the lethal temperature of tissue cells at 42 °C. This observation indicates that BPQDs-DOX@OPM demonstrated excellent in vivo photothermal performance. The temperature elevation in the tumor area of the BPQDs-DOX@OPM group can be attributed to the combined effect of effective tumor targeting and the high stability of BPQDs.

Fig. 7
figure 7

BPQDs-DOX@OPM in vivo temperature rise curves. a NS, BPQDs, BPQDs-DOX@OPM In vivo thermal imaging photography; b NS, BPQDs, BPQDs-DOX@OPM In vivo temperature rise curves

In vivo anti-tumor effect of BPQDs-DOX@OPM combined drug delivery system

As shown in Fig. 8a–c, the in vivo therapeutic effects of different treatment groups on OS were obviously different. Compared with the NS group, the BPQDs group and BPQDs@OPM group significantly reduced the rate of tumor volume increase. This is due to the good therapeutic effect of PTT on local tumor growth. The tumor volume in the BPQDs@OPM group was significantly smaller (p < 0.05) than in the BPQDs group. This was because the surface modification of OPM significantly improved the stability and slowed down the oxidative degradation of BPQDs; this was consistent with our preliminary assumption. The tumor growth rate inhibition was more evident in the BPQDs-DOX group than in the BPQDs and DOX groups, indicating that combined chemotherapy with PTT has more obvious advantages than a single-agent therapy for tumor treatment. However, BPQDs-DOX may be cleared by the immune system and renal excretory system in vivo owing to the immune clearance system and small size. In addition, BPQDs undergo further degradation upon exposure to oxygen and water, leading to reduced efficacy. Our BPQDs-DOX@OPM combined treatment group showed the most pronounced tumor growth inhibition at the end of the treatment.

Fig. 8
figure 8

Anti-tumor effects of BPQDs-DOX@OPM in vivo. a Tumor tissues after 18 days of intravenous injection of NS, BPQDs, DOX, BPQDs-@OPM, BPQDs-DOX, and BPQDs-DOX@OPM. b Measured tumor tissue weights after 18 days of treatment with NS, BPQDs, DOX, BPQDs-@OPM, BPQDs-DOX, and BPQDs-DOX@OPM. c Tumor volume change curves during NS, BPQDs, DOX, BPQDs-@OPM, BPQDs-DOX, and BPQDs-DOX@OPM treatment. d H&E stained images of tumor (size: 100 μm; Black arrows: aggregated tumor cell growth; Red arrows: tumor tissue necrosis; Green arrows: inflammatory cell infiltration.)

H&E staining of tumor tissue

Figure 8d illustrates the H&E staining of tumor tissues after the treatment. In the NS group, tumor cells did not exhibit significant necrosis and displayed varying cell sizes, noticeable nuclear heterogeneity, and disordered arrangement. In contrast, the remaining treatment groups exhibited tumor necrosis, intensified nuclear staining, and infiltration of inflammatory cells. Notably, the BPQDs-DOX@OPM group demonstrated extensive tumor cell necrosis, consolidation of nuclei with deepened staining, and sparse arrangement of tumor cells without nuclear schizogony. Moreover, in the BPQDs-DOX@OPM group, there was widespread tumor cell necrosis, deepening of nuclear solidification staining, and infiltration of inflammatory cells in the residual tumor cells.

In vivo biosafety studies of BPQDs-DOX@OPM combined drug delivery system

The toxic effects of nano-drug delivery systems on major organs and the whole system are considered an essential direction for assessing drug toxicity, and one of their leading indicators is a change in body weight [29]. As shown in Fig. 9a, the trend of body weight change in mice was recorded during treatment, and no significant decrease in body weight was observed in the BPQDs-DOX@OPM and BPQDs@OPM treatment groups. After the end of BPQDs-DOX@OPM in vivo treatment, the heart, liver, spleen, lungs, and kidneys of the sampled mice were dissected. As shown in Fig. 9b, the H&E staining results indicated that the BPQDs-DOX@ OPM group showed no significant damage to all organs, indicating that the BPQDs-DOX@OPM combined drug delivery system had high biosafety.

Fig. 9
figure 9

In vivo biosafety assessment of BPQDs-DOX@OPM. a H&E stained sections of each major organ of animals 18 days after intravenous injection of NS, BPQDs, DOX, BPQDs-@OPM, BPQDs-DOX, and BPQDs-DOX@OPM. b Body weight changes of animals in each group during NS, BPQDs, DOX, BPQDs-@OPM, BPQDs-DOX, and BPQDs-DOX@OPM treatment. (size: 100 μm)