Biotin conjugates in targeted drug delivery: is it mediated by a biotin transporter, a yet to be identified receptor, or (an)other unknown mechanism(s)?

Figures & data

Figure 1. Chemical structures of α-lipoic acid, pantothenic acid, biotin, and its structural analogs.

Figure 2. A proposed mechanism of SMVT-mediated uptake of biotin by a mammalian cell.

Table 1. Uptake studies of biotin in various cell lines.

Entry	Cells	Substrates used in the competition assay		Effect on biotin transport by SMVT	Conclusion	Ref.
1	Human intestinal tissue (brush-border membrane vesicle)	Biotin	Desthiobiotin	Biotin (5 µM) uptake reduced from 9.80 ± 0.60 pmol.mg protein^–1.20 s^–1 (control group) to 5.25 ± 0.18 pmol.mg protein^–1.20 s^–1 and 4.88 ± 0.14 pmol.mg protein^–1.20 s^–1 in the presence of 20 and 50 µM of desthiobiotin, respectively.	These structural analogs of biotin reduce biotin transport by SMVT at 20–50 mM in the competition assay. The absence of tetrahydroimidazolone ring (α-lipoic acid) or tetrahydrothiophene (desthiobiotin) in the structure of biotin does not significantly alter their interaction with the SMVT.	91
			α-Lipoic acid	Biotin (5 µM) uptake reduced from 9.80 ± 0.60 pmol.mg protein^–1.20 s^–1 (control group) to be 5.60 ± 0.22 pmol.mg protein^–1.20 s^–1 and 4.70 ± 0.27 pmol.mg protein^–1.20 s^–1 in the presence of 20 and 50 µM of α-lipoic acid, respectively.
			Biotin methyl ester	Biotin (5 µM) uptake reduced slightly from 9.80 ± 0.60 pmol.mg protein^–1.20 s^–1 (control group) to 8.37 ± 0.33 pmol.mg protein^–1.20 s^–1 and 7.95 ± 0.34 pmol.mg protein^–1.20 s^–1 in the presence of 20 and 50 µM of biotin methyl ester, respectively.	Biotin methyl ester and α-lipoic amide having modified carboxylic acid group and do not effectively compete with the process of biotin transport by SMVT.
			α-Lipoic amide	Biotin (5 µM) uptake slightly reduced from 9.80 ± 0.60 pmol.mg protein^–1.20 s^–1 (control group) to 9.10 ± 0.44 pmol.mg protein^–1.20 s^–1 in the presence of 50 µM of α-lipoic amide.
2	Human-derived colon cell line, Caco-2 cells	[^3H]biotin	Unlabelled biotin	The transport of [^3H]biotin (4 nM) reduced from 1.69 ± 0.04 pmol.mg protein^–1.3 min^–1 to 0.664 ± 0.03 pmol.mg protein^–1.3 min^–1 and 0.56 ± 0.02 pmol.mg protein^–1.3 min^–1 in the presence of 25 and 50 µM of unlabelled biotin, respectively.	Co-treatment with unlabelled biotin and desthiobiotin led to inhibition of the transport of [^3H]biotin. This study establishes the critical role of the carboxylic acid group in the biotin molecule for its transport by SMVT.	88
			Desthiobiotin	The transport of [^3H]biotin (4 nM) reduced from 1.69 ± 0.04 pmol.mg protein^–1.3 min^–1 to 0.95 ± 0.02 pmol.mg protein^–1.3 min^–1 and 0.73 ± 0.01 pmol.mg protein^–1.3 min^–1 in the presence of 25 and 50 µM of desthiobiotin, respectively.
			Biocytin	The uptake of [^3H]biotin (4 nM) reduced from 1.69 ± 0.04 pmol.mg protein^–1.3 min^–1 to 1.47 ± 0.12 pmol.mg protein^–1.3 min^–1 and 1.16 ± 0.05 pmol.mg protein^–1.3 min^–1 in the presence of 25 and 50 µM of biocytin, respectively.	Biotin analogs like biocytin and biotin methyl ester having modified carboxylic acid group and had a non-significant effect on [^3H]biotin transport by SMVT. While tetrahydroimidazolone ring (absent in diaminobiotin) fused with tetrahydrothiophene ring also seems to be important for interaction with SMVT.
			Biotin methyl ester	25 and 50 µM concentrations of biotin methyl ester and diaminobiotin had a non-significant effect on [^3H]biotin (4 nM) uptake.
			Diaminobiotin
3	Kidney cells (MDCK-MDR1)	[^3H]biotin	Unlabelled biotin	Compared to the control, the uptake of [^3H]biotin (10 nM) significantly reduced to 9.03 ± 1.51%, 51.50 ± 7.46%, and 16.27 ± 0.47% in the presence of 1 mM, 10 µM, and 100 µM of unlabelled biotin, respectively.	Data analysis showed that the unlabelled biotin, pantothenic acid, lipoic acid, valeric acid, and desthiobiotin significantly inhibited the uptake of [^3H]biotin in the presence of varying concentrations. While biotin analogs with a modified carboxylic acid such as biotin methyl ester and biocytin do not effectively compete with the uptake of [^3H]biotin.	33
			Pantothenic acid	Compared to the control, the uptake of [^3H]biotin (10 nM) significantly reduced to 9.12 ± 1.39%, 27.22 ± 1.06%, and 25.62 ± 2.06% in the presence of 1 mM, 10 µM, and 100 µM of pantothenic acid, respectively.
			Desthiobiotin	Compared to the control, the uptake of [^3H]biotin (10 nM) significantly reduced to 15.61 ± 2.41%, 40.97 ± 4.29%, and 16.67 ± 1.69% in the presence of 1 mM, 10 µM, and 100 µM of desthiobiotin, respectively.
			α-Lipoic acid	Compared to the control, the uptake of [^3H]biotin significantly reduced to 6.97 ± 0.82% in the presence of 1 mM of α-lipoic acid.
			Valeric acid	The uptake of [^3H]biotin was significantly reduced to 60.39 ± 4.02% in the presence of 1 mM of valeric acid, compared to the control.
4	Human-derived retinoblastoma cell line (Y-79)	[^3H]biotin	Unlabelled biotin	The uptake of [^3H]biotin (10 nM) significantly decreased to 45.2% and 73.6% in the presence of 50 µM and 100 µM of unlabelled biotin, respectively when compared to the control group.	Competition with unlabelled biotin, lipoic acid, pantothenic acid, desthiobiotin, and valeric acid significantly decreased the uptake of [^3H]biotin at various concentrations. While the biotin analogs, such as biocytin and biotin methyl ester were non-significant to decrease the uptake of [^3H]biotin.	93
			α-Lipoic acid	The uptake of [^3H]biotin (10 nM) significantly decreased to 53.9% and 74.1% in the presence of 50 µM and 100 µM of lipoic acid, respectively, when compared to the control group.
			Pantothenic acid	The uptake of [^3H]biotin (10 nM) significantly decreased to 39.2% and 65.6% in the presence of 50 µM and 100 µM of pantothenic acid, respectively as compared to the control group.
			Desthiobiotin	Compared to the control group, the uptake of [^3H]biotin (10 nM) significantly decreased to ∼35% and ∼65% at 50 and 100 µM of desthiobiotin, respectively.
			Valeric acid	Compared to the control group, the uptake of [^3H]biotin (10 nM) significantly decreased to 38.6% at 100 µM of valeric acid.
5	Breast cancer cells (T47D)	[^3H] biotin	Unlabelled biotin	Compared to the control group, the uptake of [^3H]biotin (8.3 nM) significantly reduced to 49.24 ± 3.48%, 22.41 ± 3.31%, and 20.74 ± 2.17% in the presence of 10, 50, and 100 µM of unlabelled biotin, respectively.	This study corroborates the strong competitive effect of unlabelled biotin, desthiobiotin, pantothenic acid, and lipoic acid on the uptake of 8.3 nM [^3H]biotin in a concentration-dependent manner.	89
			Desthiobiotin	Compared to the control group, the uptake of [^3H]biotin (8.3 nM) significantly reduced to 62.44 ± 2.25%, 36.1 ± 3.59%, and 22.33 ± 1.05% in the presence of 10, 50, and 100 µM of desthiobiotin, respectively.
			Biocytin	Compared to the control group, the uptake of [^3H]biotin (8.3 nM) decreased to 90.71 ± 11.70%, 79.25 ± 24.16%, and 76.56 ± 21.99% in the presence of 10, 50, and 100 µM of biocytin, respectively.	Biocytin produced a weak inhibitory effect on the uptake of [^3H]biotin.
			NHS–biotin	Compared to the control group, the uptake of [^3H]biotin (8.3 nM) decreased to 60.75 ± 4.45%, 38.41 ± 10.71%, and 28.17 ± 10.77% at 10, 50, and 100 µM, respectively.	As NHS–biotin is a reactive compound, there is a high probability that it could acylate the cell surface proteins or get hydrolysed in the culture medium under experimental conditions ^115. In such a case, the hydrolysed product would effectively compete with the [^3H]biotin for uptake by SMVT.

Figure 3. A general representation of SAR of SMVT substrates.

Figure 4. Chemical structures of the biotin-conjugates described in Table 2.

Table 2. Applications of biotin in the development of drugs and diagnostics.

Entry	Application	Model	Biotin conjugate	Results	Ref.
1	Cancer diagnosis	A549 cells, in vitro	Coumarin (compound 1)	In cell imaging experiments, biotin-appended coumarin conjugate (5 µM) showed strong fluorescence within 20 min of incubation in the cells compared to the compound without biotin. Hence, compound 1 is said to show the potential to be used as cancer-selective imaging probe.	121
2	Cancer-targeting theranostic	A549 cells, in vitro	Gemcitabine (compound 2)	In a cell viability assay, compared to the untreated group, biotin-conjugated gemcitabine (1 µM) reduced the cell viability to <20% while the cell viability was found to be slightly >70% when treated with gemcitabine (1 µM) alone for 72 h.	123
3	Cancer-targeting theranostic	HeLa cells, in vitro	SN-38 (compound 3)	At 10 µM, the cell viability after treatment SN-38 (alone) and biotin-conjugated SN-38 were found to be ∼20% and ∼40% after 48 h incubation, respectively.	80
4	Tumour-targeting drug delivery	L1210F cells, in vitro	Paclitaxel derivative SB-T-1214 (compound 4)	Biotin-conjugated SB-T-1214 resulted in a marginal reduction of IC50 value (8.8 nM) compared to the non-conjugated compound (9.5 nM) after 72 h incubation as shown by the MTT assay.	46
5	Cancer-targeted drug delivery	PC-3, SGC7901, SGC7901/CDDP cells, in vitro	Cisplatin-indomethacin hybrid (compound 5)	1–3-fold improved cellular accumulation demonstrated by the biotin conjugate over the parent drug alone as determined by the measurement of the intracellular cisplatin content using ICP-MS after 12 h of incubation.	124
6	Drug delivery	Gene-knocked out diabetic (SLAC/GK) rats, in vivo	Insulin	Compared to the original blood glucose concentration, the maximum reduction of blood glucose level was found to be 56% and 70% after 20 IU/kg dose administration of biotinylated and non-biotinylated liposomes, respectively. Such result is considered proof of concept that biotinylated liposomes can be employed as carriers for oral drug delivery.	125
7	Drug delivery	Caco-2 cells, in vitro	Salmon calcitonin	A 2.5- and 4-fold increase in apical-to-basolateral permeability was demonstrated by mono- and di-biotinylated conjugates, respectively, as opposed to the non-biotin conjugated form. Similar to the entry 6, this study is said to show the potential of biotinylated carriers for improving oral drug delivery.	83
8	Cancer-targeted drug delivery	MDA-MB-436 cells, in vitro	Curcumin contained in poly (ethylene glycol)–poly (epsilon-caprolactone) micelles	The cellular uptake of biotinylated micelles showed a 4-fold improvement over its non-biotinylated micelles, as shown by their mean fluorescence density values after 2 h incubation.	126
9	Cancer-targeted drug delivery	P815 cells, in vitro	Squamocin (compound 6)	Biotin-conjugated squamocin resulted in the reduction of IC50 value (0.75 ± 0.02 µM) compared to the squamocin alone (19.80 ± 1.18 µM) after 72 h incubation as shown by the MTT assay.	127
10	Cancer-targeting theranostic	HeLa cells, in vitro	Paclitaxel surface-functionalised on gold nanoparticle (AuNP)	Biotin conjugate AuNP showed >2-fold improvement in cytotoxicity in comparison to its non-biotin conjugated version after 24 h incubation.	128

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