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

Abstract

Biotin molecules could be used as suitable targeting moieties in targeted drug delivery systems against tumors. To develop a biotin targeted drug delivery system, we employed human serum albumin (HSA) as a carrier. Methotrexate (MTX) molecules were conjugated to HSA. MTX-HSA nanoparticles (MTX-HSA NPs) were prepared from these conjugates by cross-linking the HSA molecules. Biotin molecules were then conjugated on the surface of MTX-HSA NPs. The anticancer efficacy of biotin targeted MTX-HSA NPs was evaluated in mice bearing 4T1 breast carcinoma. A single dose of biotin targeted MTX-HSA NPs showed stronger in vivo antitumor activity than non-targeted MTX-HSA NPs and free MTX. By 7 days after treatment, average tumor volume in the biotin targeted MTX-HSA NPs-treated group decreased to 17.6% of the initial tumor volume when the number of attached biotin molecules on MTX-HSA-NPs was the highest. Average tumor volume in non-targeted MTX-HSA NPs-treated mice grew rapidly and reached 250.7% of the initial tumor volume. Biotin targeted MTX-HSA NPs increased the survival of tumor-bearing mice to 47.5 ± 0.71 days and increased their life span up to 216.7%. Mice treated with biotin targeted MTX-HSA NPs showed slight body weight loss (8%) 21 days after treatment, whereas non-targeted MTX-HSA NPs treatment at the same dose caused a body weight loss of 27.05% ± 3.1%.

Keywords: biotin; conjugates; human serum albumin; in vivo anticancer delivery; methotrexate; targeted drug delivery.

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.

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.