Use of biotin targeted methotrexate–human serum albumin conjugated nanoparticles to enhance methotrexate antitumor efficacy

Figures & data

Table 1 Physico-chemical characteristics of MTX-HSA NPs (MTX/HSA molar ratio = 8) and biotin functionalized MTX-HSA NPs (mean ± standard deviation; n = 3)

Biotin/HSA molar ratio	MTX-HSA NPs	Biotin 3.66-MTX-HSA NPs	Biotin 7.01-MTX-HSA NPs	Biotin 9.41-MTX-HSA NPs
	0	3.66 ± 0.22	7.01 ± 0.14	9.41 ± 0.34
Particle size (nm)	111.70 ± 4.60	125.10 ±3.40	132.40 ± 2.14	144.36 ± 3.80
Poly dispersity index	0.10 ± 0.01	0.21 ± 0.07	0.19 ± 0.04	0.24 ± 0.05
Zeta potential (mV)	−12.10 ± 0.5	−20.23 ± 1.11	−19.56 ± 2.01	−21.54 ± 1.25

Abbreviations: MTX-HSA NPs, methotrexate–human serum albumin conjugated nanoparticles; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratio; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate-human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/ human serum albumin molar ratio.

Figure 1 Antitumor effect of free MTX, MTX-HSA NPs, and biotin 7.01-MTX-HSA NPs on 4T1 tumor-bearing mice. 4T1 tumor cells were implanted subcutaneously in Balb/c mice. The drugs were injected intravenously in a single dose (day 0). The doses were equivalent to 6.25 and 12.5 mg/kg of free MTX. Data are presented as mean ± standard deviation of relative tumor volumes (day 0 taken as 100%).

Abbreviations: MTX, methotrexate; MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio.

Figure 2 Antitumor effect of MTX-HSA NPs, biotin 3.66-MTX-HSA NPs, biotin 7.01-MTX-HSA NPs, and biotin 9.41-MTX-HSA NPs on 4T1 tumor-bearing mice. 4T1 tumor cells were implanted subcutaneously in Balb/c mice. The drugs were injected intravenously in a single dose (day 0). The doses were equivalent to 12.5 mg/kg of free MTX. Data are presented as mean ± standard deviation of relative tumor volumes (day 0 taken as 100%).

Abbreviations: MTX, methotrexate; MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratio; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/human serum albumin molar ratio.

Table 2 Effect of biotin targeting on the survival of 4T1 tumor-bearing mice

Group	Median survival time (day)	Life span increase (%)
Saline	15.01 ± 0.79	–
MTX	17.51 ± 0.60	16.66
MTX-HSA NPs	22.52 ± 0.50	50.10
Biotin 3.66-MTX-HSA NPs	32.5 ± 0.41	116.66
Biotin 7.01-MTX-HSA NPs	37.5 ± 0.67	150.10
Biotin 9.41-MTX-HSA NPs	47.5 ± 0.71	216.66

* Note: The dose was equivalent to 6.25 mg/kg of free MTX.

Abbreviations: MTX, methotrexate; MTX-HSA NPs, methotrexate–human serum albumin conjugated nanoparticles; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratios; biotin 7.02-MTX-HSA NPs, biotin targeted methotrexate– human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/human serum albumin molar ratio.

Figure 3 Animal survival study. The 4T1 tumor-bearing mice were treated with free MTX, MTX-HSA NPs, biotin 3.66-MTX-HSA NPs, biotin 7.02-MTX-HSA NPs, and biotin 9.41-MTX-HSA NPs. The drugs were injected intravenously in a single dose (day 0). The doses were equivalent to 12.5 mg/kg of free MTX. The curve shows the number of 4T1 tumor-bearing mice still alive on different days.

Abbreviations: MTX, methotrexate; MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratio; biotin 7.02-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratios; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/human serum albumin molar ratio.

Figure 4 Change in body weight of 4T1 tumor-bearing mice treated with free MTX, MTX-HSA NPs, biotin 3.66-MTX-HSA NPs, biotin 7.01-MTX-HSA NPs, and biotin 9.41-MTX-HSA NPs. The drugs were injected intravenously in a single dose (day 0). The doses were equivalent to 6.25 mg/kg of free MTX.

Abbreviations: MTX, methotrexate; MTX-HSA NPs, methotrexate–human serum albumin conjugated nanoparticles; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratio; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratios; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/human serum albumin molar ratios.

Figure S1 Transmission electron microscopy image of biotin 7.01-MTX-HSA NPs.

Abbreviations: MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio.

Figure S2 Storage stability of biotin 7.01-MTX-HSA NPs. Size and zeta potential of NPs were recorded for 6 weeks.

Abbreviations: NP, nanoparticle; MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate– human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio.

Figure S3 The uptake of FITC-labeled non-targeted MTX-HSA NPs (A) biotin 3.66-MTX-HSA NPs (B), biotin 7.01-MTX-HSA NPs (C), biotin 9.41-MTX-HSA NPs (D) at concentration of 1 mg/mL to T47D tumor cells after 4 hours at 37°C. The uptake of FITC-labeled non-targeted MTX-HSA NPs (E), biotin 3.66-MTX-HSA NPs (F), biotin 7.01-MTX-HSA NPs (G), biotin 9.41-MTX-HSA NPs (h) at concentration of 1 mg/mL to HeLa tumor cells after 4 hours at 37°C.

Abbreviations: FITC, fluorescein isothiocyanate; MTX, methotrexate; MTX-HSA NP, methotrexate–human serum albumin conjugated nanoparticle; biotin 3.66-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 3.66 ± 0.22 biotin/human serum albumin molar ratio; biotin 7.01-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 7.01 ± 0.14 biotin/human serum albumin molar ratio; biotin 9.41-MTX-HSA NPs, biotin targeted methotrexate–human serum albumin nanoparticles: 9.41 ± 0.34 biotin/human serum albumin molar ratio.