Introduction

Cutaneous melanoma is a common skin cancer with high degree of malignancy, its formation has a close relationship with excessive ultraviolet (UV) radiation1,2. Because of through long-term and lots of high-intensity exercise training outdoors, athletes in outdoor sports become a high-risk group for CM due to overexposure of UV radiation3,4. For example, marathoners, yachtsmen, triathletes, baseball players, footballers, tennis players and so on5,6. The most expected therapeutic effect of outdoor athletes suffering from CM is painlessness, no surgical trauma, no physical adverse reactions and high efficiency, the treatment process is easy to operate, ensuring that their outdoor training plans and competition goals can be successfully achieved. However, the conventional treatments will damage the function of the human motor system to varying degrees, resulting in persistent adverse physical reactions or (and) pain, which has serious impacts on the sports career of most athletes suffering from CM. Chemotherapy is considered as effective treatment to curb the development of CM, but its grievous side effects often lead to impaired or even lost athletic ability in athletes. For example, the nonspecific distribution of chemotherapeutics always damage the normal tissues. Radiotherapy may cause poor effect due to strong resistance of CM to ionizing radiation. Surgery usually injure the adjacent tissues7,8. Therefore, safe and effective treatment is particularly important for outdoor athletes suffering from CM.

The application of nanotechnology in drug delivery is a hopeful method to both for achieving satisfactory curative effect and retaining athletes’ motor abilities in CM treatment. On account of that nanocarriers can encapsulate anticancer drugs and specifically deliver them to tumor tissues and cells based on the enhanced permeability and retention (EPR) effect of tumor tissues and the various tumor-targeting ligands (such as aptamers, antibodies, peptides and some other small molecules) coupled on the surface of nanocarriers which can conjugated to specific receptors overexpressed on the membrane surface of cancer cells9,10. So the distribution of anticancer durgs loaded in nanocarriers in normal tissues is decreased dramatically that the side effects of drugs are significantly relieved while the anti-tumor efficacy is enhanced11,12,13. Up to now, nanocarrier of anticancer drugs was obtained some promising research results in the therapies of liver cancer, lung cancer, pancreatic cancer and so on14,15,16. The drug-loaded nanocarriers have also been certified to effectively inhibit the progression of CM and improve the overall survival rate of patients10,17. However, the therapeutic effect achieved by tumor targeted nanocarriers carrying a single drug was invariably hampered by multidrug resistance (MDR) of CM cells. Therefore the combined targeted treatment carrying two drugs at the same time, aimed at specifically killing CM cells and preventing their MDR, had attracted the attention of experts and relevant researches had been carried out18,19.

Chemotherapy drugs usually have good performance in killing CM cells, such as doxorubicin (DOX) and paclitaxel (PTX)20,21. Small interfering RNA (siRNA) can effectively and specifically inhibit the expression of target proteins related to MDR in cancer cells, for example growth factors and drug efflux pumps22,23. The combination of chemotherapeutics and siRNA separately in the treatment of CM has been exerted positive effects24. However, co-delivery of chemotherapeutics and siRNA to CM tissue would be more efficient in both killing CM cells and inhibiting their MDR. Due to the completely different physicochemical properties and in vivo pharmacokinetic behaviors of chemotherapeutics and siRNA, the design and preparation of nanocarrier for the co-delivery of these two drugs are facing serious challenge. Previously there were few reports on the co-delivery nanocarrier of chemotherapeutics and siRNA, but relevant researches had indicated their valuable application prospects25.

In our previous study26,27, a novel tumor-targeted co-carrier of anti-cancer drugs and siRNA was designed and prepared. Firstly, the new amphiphilic grafted polymer, namely folate–biotin-quaternized starch (FBqS), was synthesized by coupling folate (as target ligand) and biotin (as hydrophobic ligand) to main chain of quaternized starch via ester condensation. The amphiphilic FBqS polymers could self-assemble to form core/shell spherical nanoparticles (FBqS NPs) in aqueous solution, hydrophobic small molecular chemotherapeutics could be encapsulated in the core by hydrophobic interaction and negatively charged siRNA could be loaded on the cationic hydrophilic shell by electrostatic adsorption. The folate (FA) on the shell of NPs serves as tumor-targeting ligand because of its specific binding ability to folate receptor overexpressed on the surface of majority cancer cells, so that FBqS NPs can achieve the tumor targeted co-delivery of chemotherapeutics and siRNA. DOX and siRNAIGF1R (IGF1R, Insulin-like growth factor 1 receptor) were used as model hydrophobic chemotherapeutics and MDR inhibition-related siRNA in our research. The physicochemical properties, co-carrier characteristics, blood compatibility and serum stability of drugs (DOX and siRNAIGF1R) loaded, drugs loading capacities and release behaviors of FBqS NPs were all determined carefully (A detailed synthesis procedure and the characterization of physicochemical properties can be found in the Supplementary Material). The results showed that FBqS NPs were superior co-carrier of chemotherapeutics and siRNA, and have promising application prospect in tumor therapy. Then, we had treated A549 cells (human lung adenocarcinoma cell lines) by siRNAIGF1R/DOX/FBqS NPs to evaluate the therapeutic effect on human lung cancer. The result indicated that the blank FBqS NPs showed biosafety to A549 cells. Compared with siRNAIGF1R/FBqS NPs and DOX/FBqS NPs loaded with single drug, siRNAIGF1R/DOX/FBqS NPs exhibited the strongest cytotoxicity and inhibitory effect on cell proliferation.

These previous works promoted us to further explore the potential application of FBqS NPs as the targeted co-delivery platform for chemotherapeutics and gene drugs in CM treatment. In the present study, A375 cells (human malignant melanoma cell lines) were cultured with siRNAIGF1R/DOX/FBqS NPs, and their intracellular distribution, cytotoxicity, inhibition of cell proliferation and migration, competition for targeted ligand, mechanism of cellular uptake and endocytosis, and downregulation of target protein expression were determined specifically. The research results were compared with the effects of free DOX, DOX/FBqS NPs, and siRNAIGF1R/FBqS NPs.

Materials and methods

Materials and cell culture

The blank, various drug-loaded biotin-quaternized starch nanoparticles (BqS NPs) and FBqS NPs in different drug concentrations were prepared according to our previous work26. All the other reagents used in this study were described earlier27. A375 cells were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cells were incubated in humidified atmosphere (containing 5% CO2) incubator at 37 °C. The cells in the logarithmic growth phase determined by cell counting were used in all experiments and the experiments were independently performed at least three times.

Cytotoxicity

The cytotoxicity of free DOX, blank and drug-loaded FBqS NPs to A375 cells was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry (FCM).

For MTT assay, A375 cells were seeded in 96-well plate at the density of 5 × 103 cells/well and incubated overnight, then the attached cells were cultured in fresh medium containing separately blank FBqS NPs, siRNAIGF1R/FBqS NPs, free DOX, DOX/FBqS NPs or siRNAIGF1R/DOX/FBqS NPs at the same concentrations of FBqS NPs, siRNAIGF1R and DOX. The concentration ranges of FBqS NPs, siRNAIGF1R and DOX were severally from 13.3–106.6 mg/l, 0.3–2.4 mg/l and 1–8 mg/l (Supplementary Table S1). The untreated cells were used as blank control and every sample was repeated in 5 different wells. After 48 h of incubation, the cells were washed twice with cold PBS and continuously cultured in fresh medium containing MTT reagent (0.5 mg/ml) for 4 h. Afterwards, the culture mediums were removed and 100 μl DMSO was added in each well to dissolve the formazan crystals. The percentage of cell viability relative to the control sample was estimated by a microplate reader (TECAN Spark, Switzerland) at the wavelength of 570 nm. The synergistic effect of DOX and siRNAIGF1R exerted by siRNAIGF1R/DOX/FBqS NPs on A375 cells was measured from the MTT results of siRNAIGF1R/FBqS NPs, DOX/FBqS NPs and siRNAIGF1R/DOX/FBqS NPs by Compusyn software based on Chou-Talalay method.

For FCM assay, A375 cells were seeded in 6-well plate at the density of 5 × 105 cells/well and incubated overnight, then cells were respectively cultured in fresh mediums containing separately blank FBqS NPs, siRNAIGF1R/FBqS NPs, free DOX, DOX/FBqS NPs or siRNAIGF1R/DOX/FBqS NPs with equal concentrations of 3 mg/l DOX, 0.9 mg/l siRNAIGF1R and 40 mg/l FBqS NPs. The control cells were cultured without any treatment. After incubation for 48 h, all A375 cells were washed, harvested and centrifuged for purification, then the cells were suspended again and stained by Annexin V-FITC. The apoptosis of samples was determined by FCM using a BD LSRFortessa (BD Biosciences, USA).

Wound healing assay

Cell scratch test was applied to show the impact of drugs on inhibiting the proliferation and migration of A375 cells. Briefly, A375 cells were seeded in dishes of 35 mm diameter at the density of 5 × 105 cells/dish. After incubation for 12 h, a 200 μl sterile pipette tip was used to form a linear scratch of the same width wound on the cell monolayer attached and converged in every dish. Then, cell debris was washed away from dishes with cold PBS. The scratched cell monolayers were separately incubated in fresh culture mediums containing constant concentration of free DOX, DOX/BqS NPs, DOX/FBqS NPs or siRNAIGF1R/DOX/FBqS NPs (DOX: 2 mg/l, siRNAIGF1R: 0.6 mg/l) for 48 h. The scratched cell monolayer was incubated in fresh culture medium without any drugs as the control sample. After incubation, all samples were washed and fixed with 4% paraformaldehyde and the wound width of every sample was observed on an inverted microscope (Olympus IX7, Japan), wound healing rate was calculated according to the change in wound width before and after drug treatment27.

Western blotting

The influence of siRNAIGF1R/FBqS NPs on IGF1R protein expression in A375 cells was estimated by western blotting. Briefly, cells were seeded in 6-well plate (5 × 105 cells/well) and cultured overnight, then cells were severally incubated in fresh culture medium containing two doses of siRNAIGF1R/FBqS NPs (siRNAIGF1R: 1 or 2 mg/l) for 48 h with the cells without any drug treatment as blank control. After incubation, the cells were washed, collected and lysed. The cellular protein was extracted from lysate and protein concentration was measured by BCA protein assay kit. The experimental procedure of Western blotting assay was the same as previously described27 and protein bands were imaged and analyzed by Chemiluminescent Imaging System (Tanon 5200, Shanghai).

Free folate competition

Folate receptor was overexpressed on the membrane of A375 cell according to the previous reports28, the free folate competitive inhibition on cell uptake of siRNAIGF1R/DOX/FBqS NPs was researched through MTT assay and confocal laser scanning microscopy (CLSM) image.

For MTT assay, A375 cells were seeded in 96-well plate (5 × 103 cells/well) and incubated overnight to facilitate cell adhesion. Then, the original culture mediums were replaced with fresh culture mediums containing with constant concentration of siRNAIGF1R/DOX/FBqS NPs (DOX: 2 mg/l, siRNAIGF1R: 0.6 mg/l) and various concentrations of free folate (from 0 to 500 mg/l), samples were incubated for 48 h. The rest processes of experiment were the same as the MTT assay described in “Cytotoxicity” section.

For CLSM image, A375 cells were seeded in confocal dishes at the density of 5 × 105 cells/dish and incubated to attached. Next, cells were incubated in fresh culture medium containing constant concentration of FAM-siRNAIGF1R/DOX/FBqS NPs, FAM-siRNAIGF1R/DOX/BqS NPs or free DOX (DOX: 2 mg/l, siRNAIGF1R: 0.6 mg/l, FAM: carboxyfluorescein) with or without free folate (100 mg/l). The incubation lasted 4 h, then sample cells were washed, fixed and labeled nuclei with Hoechst 33342 (blue fluorescence). CLSM (Olympus FV1000, Japan) was used to observe the uptake of DOX (red fluorescence) and FAM-siRNAIGF1R (green fluorescence) by A375 cells.

Cellular uptake and distribution

The characteristics of cellular uptake and intracellular distribution of siRNAIGF1R/DOX/FBqS NPs and free DOX in A375 cells were observed by CLSM. Cells were plated in confocal dishes (5 × 105 cells/dish) before the test. After attached, they were independently cultured with FAM-siRNAIGF1R/DOX/FBqS NPs or free DOX comprised equal concentration of 3 mg/l DOX, and 0.9 mg/l siRNAIGF1R for different time intervals (30 min, 1 h, 4 h). Then, the next steps of experiment were the same as the CLSM image described in “Free folate competition” section.

Endocytosis mechanism survey

The endocytosis mechanism of siRNAIGF1R/DOX/FBqS NPs in A375 cells was detected by CLSM image and FCM. Several specific endocytosis inhibitors of sodium azide (NaN3: energy inhibitor), colchicine (COLC: microtubule-dependent macropinocytosis inhibitor), chlorpromazine (CPZ: clathrin-mediated endocytosis inhibitor), indomethacin (INDO: caveolae-mediated endocytosis inhibitor), folate (FA: folate-receptor competitive inhibitor) were used to screen the cellular endocytic pathway.

For CLSM, the A375 cells attached in confocal dishes (5 × 105 cells/dish) were per-incubated in culture medium respectively containing NaN3 (15 mg/l), COLC (50 mg/l), CPZ (10 mg/l), INDO (50 mg/l) or FA (300 mg/l) for 1 h. Then cells were cultured in fresh culture medium containing FAM-siRNAIGF1R/DOX/FBqS NPs (DOX: 2 mg/l, siRNAIGF1R: 0.6 mg/l) for 4 h. The following experimental steps were the same as the CLSM image described in “Free folate competition” section.

For FCM, the A375 cells attached in 6-well plate (5 × 105 cells/dish) were incubated with the above endocytosis inhibitors separately for 1 h and next FAM-siRNAIGF1R/DOX/FBqS NPs for 4 h. The positive control cells were treated only with FAM-siRNAIGF1R/DOX/FBqS NPs without any inhibitors for 4 h, while the negative control cells were incubated in normal culture medium without any treatment. After incubation, above cells were washed, harvested and centrifuged for purification, FCM was utilized to test the fluorescence intensities of FAM-siRNAIGF1R (green) and DOX (red) in suspension cells.

Statistical analyses

Based on at least three repeated experimental results, the statistical analysis was carried out by using SPSS Statistics 17.0 software (IBM, USA). All the data was shown as mean ± standard deviation (SD). The statistical differences between samples were measured by One-Way ANOVA and two-tailed Student’s t-test, *p < 0.05 was judged as statistical significance.

Results

Cytotoxicity of siRNAIGF1R/DOX/FBqS NPs

The cytotoxicity results of blank FBqS NPs, different drug-loaded FBqS NPs and free DOX at various drug concentrations against A375 cells were displayed in Fig. 1a and b. The data of MTT assay (Fig. 1a) showed that effect of blank FBqS NPs on cell viability was negligible at all various concentrations and siRNAIGF1R/FBqS NPs also exhibited hypotoxicity to A375 cells so that it’s half maximal inhibitory concentration (IC50) was not obtained even at the high concentration of 2.4 mg/l, because siRNAIGF1R/FBqS NPs did not have the ability to directly kill A375 cells. In contrast with blank FBqS NPs and siRNAIGF1R/FBqS NPs, A375 cells showed high sensitivity to free DOX (IC50: 4.53 mg/l), DOX/FBqS NPs exhibited stronger cytotoxicity (IC50: 2.11 mg/l for DOX content) due to the folate-receptor-mediated endocytosis on targeted NPs. Based on the synergistic effect of DOX and siRNAIGF1R against A375 cells, siRNAIGF1R/DOX/FBqS NPs possessed the highest cytotoxicity (IC50: 1.67 mg/l for DOX content) among all administration groups. The combination index (CI) data (Supplementary Table S2; Supplementary Fig. S5) showed that a synergistic effect occurs when the total dose of DOX and siRNAIGF1R in siRNAIGF1R/DOX/FBqS NPs is higher than or equal to 6.5 mg/l.

Fig. 1
figure 1

Cytotoxicity of FBqS NPs, siRNAIGF1R/FBqS NPs, free DOX, DOX/FBqS NPs and siRNAIGF1R/DOX/FBqS NPs against A375 cells by MTT assay (a) and FCM assay (b). The contents of FBqS NPs are about 13.3 mg/l, 26.7 mg/l, 40 mg/l, 53.3 mg/l, 66.6 mg/l, 80 mg/l, 93.3 mg/l and 106.6 mg/l, respectively corresponding to concentrations of DOX from 1 to 8 mg/l and siRNAIGF1R from 0.3 to 2.4 mg/l in (a); Wound healing images (c) and rate (d) of A375 cells incubated separately with different drug formulations at constant drugs concentrations, cells images were taken under 20× magnification; Western blotting result of IGF1R protein expression levels in A375 cells incubated with different doses of siRNAIGF1R/FBqS NPs for 48 h, β-actin protein was used as the loading control (e), original image is presented in Supplementary Fig. S4. *p < 0.05, **p < 0.01, ***p < 0.001.

Full size image

The effects on A375 cells viability of the above different drug formulations with equal NPs and (or) drug concentrations were also evaluated by FCM (Fig. 1b). The fluorescence intensities of Annexin V-FITC in various samples displayed the apoptosis in different drug formulations, and the order of Annexin V-FITC fluorescence intensity from weak to strong was blank control < FBqS NPs < siRNAIGF1R/FBqS NPs < free DOX < DOX/FBqS NPs < siRNAIGF1R/DOX/FBqS NPs. This conclusion was consistent with the results of MTT assay, siRNAIGF1R/DOX/FBqS NPs presented the strongest cytotoxicity among all drug formulations. The cytotoxicity of FBqS NPs was the weakest, close to that of the control sample.

Inhibition of A375 cell porliferation and migration

The inhibitory effects of different treatments on A375 cell proliferation and migration were observed through wound healing assay. After cell monolayer with the same width linear scratch was co-incubated with various drug formulations separately, the change in wound width of the cell monolayers were shown in Fig. 1c and d. The samples’ order of wound healing rate from high to low was control (0.76) > free DOX (0.41) > DOX/BqS NPs (0.29) > DOX/FBqS NPs (0.08) > siRNAIGF1R/DOX/FBqS NPs (− 0.08). This order indicated that siRNAIGF1R/DOX/FBqS NPs had the strongest antimigration and antiporliferation effects on A375 cells, the wound healing rate of single drug-loaded NPs was lower than that of siRNAIGF1R/DOX co-loaded NPs but higher than that of free DOX. Meanwhile, the targeted endocytosis mediated by folate receptor also significantly reduced the healing of scratched cell monolayer.

Reduction of IGF1R protein level

The downregulation of target protein expression levels in A375 cells was detected by western blotting. After treatment with different doses of siRNAIGF1R/FBqS NPs, the expression level of IGF1R protein in A375 cells was significantly reduced compared to that in the untreated sample (Fig. 1e). As the dosage of siRNAIGF1R/FBqS NPs increased, the strength ratio of two protein bands (IGF1R/β-actin) decreased from 0.72 to 0.35 in dose-dependent manner. The result indicated that siRNA was successfully delivered into the cytoplasm through FBqS NPs and effectively inhibited the expression level of target protein in A375 cells.

Free folate competitive inhibition

The folate competition assay was used to detect the role of folate ligand in the uptake of siRNAIGF1R/DOX/FBqS NPs by A375 cells. The result of MTT assay (Fig. 2a) showed cell viability was 44.5% incubated with siRNAIGF1R/DOX/FBqS NPs without free folate, but cell viability increased to 75.4% under the same culture condition containing 300 mg/l free folate. The result demonstrated free folate significantly weakened the cytotoxicity of siRNAIGF1R/DOX/FBqS NPs against A375 cells and this effect was dose-dependent.

Fig. 2
figure 2

Viability of A375 cells incubated with the same concentration of siRNAIGF1R/DOX/FBqS NPs and different concentrations of free folate (a); The distributions of FAM-siRNAIGF1R (green) and DOX (red) in A375 cells incubated with FAM-siRNAIGF1R/DOX/FBqS NPs, FAM-siRNAIGF1R/DOX/BqS NPs or free DOX at the same concentrations of FAM-siRNAIGF1R and DOX with or without excess free folate (b).

Full size image

In CLSM images (Fig. 2b), the green (FAM-siRNAIGF1R) and red (DOX) fluorescence intensities indicated the cellular uptake degree of FAM-siRNAIGF1R/DOX/FBqS NPs or FAM-siRNAIGF1R/DOX/BqS NPs in A375 cells. Under culture condition without free folate (FA), the cellular uptake of FAM-siRNAIGF1R/DOX/FBqS NPs by A375 cells was significantly higher than that under culture condition with 100 mg/l of free folate. However, under the same two cultivation conditions, there was no significant difference in the cellular uptake of FAM-siRNAIGF1R/DOX/BqS NPs by A375 cells. In addition, the cell uptake of free DOX at the same drug concentration was the least among all drug administration groups. Due to the presence of targeted folate ligand on the surface of FBqs NPs, A375 cells had the highest uptake of drug-loaded FBqs NPs. When there was competition of free folate, the uptake of drug-loaded FBqs NPs by cells significantly decreased. However, there was no targeted folate ligand on the surface of Bqs NPs, so the uptake of drug-loaded Bqs NPs by cells was relatively low, and the uptake was almost unaffected by the presence of folate. Since free DOX couldn’t enter A375 cells through endocytosis, and the free DOX was expelled from the cells due to the efflux mechanism of cells, the amount of free DOX in the cells was the lowest. The results confirmed that the folate ligand on the surface of FBqS NP could effectively enhance the endocytosis of siRNAIGF1R/DOX/FBqS NP in A375 cells, while free folate in the culture medium could competitively inhibit its endocytosis.

Cellular uptake and distribution behaviors

The cellular uptake and intracellular distribution of FAM-siRNAIGF1R/DOX/FBqS NPs or free DOX in A375 cells were explored and compared by CLSM images (Fig. 3). After incubating A375 cells with FAM-siRNAIGF1R/DOX/FBqS NPs for 30 min, a small amount of DOX and FAM-siRNAIGF1R appeared in cytoplasm. After 1 h, they grew and aggregated in cytoplasm. After 4 h of incubation, a large amount of DOX and FAM-siRNAIGF1R accumulated in cytoplasm, and many DOX had entered and even covered the cell nucleus. But for free DOX of the same drug concentration, the DOX in the cytoplasm and nucleus was much less in any equal incubation time interval. This result indicated that compared to free DOX, drug-loaded FBqS NPs significantly enhanced cellular uptake of DOX and successfully delivered siRNAIGF1R and DOX to their respective target spots in A375 cells.

Fig. 3
figure 3

CLSM images of A375 cells incubated with FAM-siRNAIGF1R/DOX/FBqS NPs or free DOX comprising equal concentrations of DOX and siRNAIGF1R for different time intervals.

Full size image

Cellular endocytosis mechanism

The CLSM images (a) and FCM measurements (b) in Fig. 4 all showed that compared to the control of without inhibitor, CPZ, INDO, and FA significantly reduced the fluorescence intensities of FAM-siRNAIGF1R and DOX in A375 cells. However, NaN3 and COLC did not significantly change the fluorescence intensities of green and red in cells. The result indicated that folate-receptor-, clathrin- and caveolae-mediated endocytosis were the main pathways for A375 cells to swallow drug-loaded FBqS NPs.

Fig. 4
figure 4

CLSM images of A375 cells incubated with FAM-siRNAIGF1R/DOX/FBqS NPs for 4 h in the presence of specific endocytosis inhibitors (a) and quantitative fluorescence intensities of DOX and FAM-siRNAIGF1R in cells were determined by FCM (b).

Full size image

Discussion

The siRNAIGF1R/DOX/FBqS NPs showed the highest cytotoxicity and proliferation suppression against A375 cells among several different formulations. The expression of IGF1R protein in A375 cell was effectively inhibited by siRNAIGF1R of targeted delivery. The presence of free folate led to a decrease in cellular uptake of siRNAIGF1R/DOX/FBqS NPs. This indicated that the drugs delivery of FBqS NPs had targeting property, specially to folate-receptor-overexpressing tumor cells. The endocytosis mediated by folate-receptor-, clathrin- and caveolae-were the prime celluar uptake pathways of drug-loaded FBqS NPs. So FBqS NPs simultaneously delivered siRNAIGF1R and DOX into A375 cells, achieving a combined and superior tumor treatment effect. Furthermore, FBqS NPs possessed the characteristics of low cytotoxicity and good biosafety.

This study is in the elementary stage of a novel drug discovery and development. Next, a series of studies (including animal experiments) need to be conducted in vivo to enrich and improve the evaluation of siRNAIGF1R/DOX/FBqS NPs for CM therapy in the future clinical application. The DOX and siRNAIGF1R can also be replaced by other chemotherapeutics and gene drugs in order to discover more potential applicabilities of FBqS NPs as a co-loading platform for targeted cancer treatment.

Conclusion

The siRNAIGF1R/DOX/FBqS NPs are expected to acquire better chemotherapy efficacy and fewer side effects on CM treatment through intravenous injection. This has significant advantages in alleviating patients’ persistent adverse physical reactions and pain, avoiding surgical trauma, and being easy to operate, which will offer the potential approach to effectively prolonging sports career of outdoor athletes suffering from CM.