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)?

Abstract Conjugation of drugs with biotin is a widely studied strategy for targeted drug delivery. The structure–activity relationship (SAR) studies through H3-biotin competition experiments conclude with the presence of a free carboxylic acid being essential for its uptake via the sodium-dependent multivitamin transporter (SMVT, the major biotin transporter). However, biotin conjugation with a payload requires modification of the carboxylic acid to an amide or ester group. Then, there is the question as to how/whether the uptake of biotin conjugates goes through the SMVT. If not, then what is the mechanism? Herein, we present known uptake mechanisms of biotin and its applications reported in the literature. We also critically analyse possible uptake mechanism(s) of biotin conjugates to address the disconnect between the results from SMVT-based SAR and “biotin-facilitated” targeted drug delivery. We believe understanding the uptake mechanism of biotin conjugates is critical for their future applications and further development.


Introduction
Even with the tremendous development in the area of drug discovery and development during the last 50 years 1 , there is still a large number of diseases that do not have effective treatment 2 .One can safely say that the levels of challenges are becoming progressively higher in dealing with the remaining problems, even after taking into consideration of new technologies and new scientific discoveries/insights. Future work is almost entirely focused on targeted therapies of various types, including modulation of activities of the molecular target(s) implicated in the relevant pathologies and/or targeted delivery based on molecular biomarkers.The latter often involves conjugation of a therapeutic molecule with a targeting ligand such as peptides 3-5 , nucleic acids 6,7 , micronutrients [8][9][10][11] , and antibodies 12,13 as well as the use of nanomaterials 14,15 to achieve the goals of targeted delivery.Such targeting ligands act as vectors and offer distinct physicochemical [16][17][18][19] and pharmacological 8,20-22 features resulting in improved pharmacodynamic and pharmacokinetic profiles of the appended drugs2 .We have a long-standing interest in developing novel drug delivery approaches including targeted drug delivery 17,[23][24][25][26][27][28][29][30] .Along this line, we have an interest in biotin-mediated targeted delivery of drugs to organs such as the kidney, colon, and the lung as well as cancer [31][32][33] .
Biotin 34 is a cofactor for enzymes collectively referred to as "biotin-dependent carboxylases," which are involved in the metabolism of amino acids, fatty acids, carbohydrates, and urea [35][36][37] .In addition, the roles of biotin also extend to the regulation of cellular processes, including gene expression and cell signalling 38 .Physiological concentrations of biotin have been reported to be in the low nanomolar range [39][40][41][42][43][44] .It has been reported that cancer cells overexpress biotin uptake system(s), which often leads to enhanced intracellular uptake of biotin 8,31,41,45 .A few examples are ovarian, lung, renal, colon, and breast cancers as well as leukaemia 8,38,46 .Conceivably, the over-expression of biotin uptake system(s) in a given type of cells provides a valuable strategy to selectively target the relevant cell types with a biotin analogue that are recognised by these "uptake system(s)".The very first example of "biotin-mediated" drug delivery with the aim of targeting the biotin uptake system was published in 1990 describing work in plant cells (soybean) 47 .Along this line, it should be noted that earlier work using biotin-avidin/streptavidin interactions for drug delivery was not intended to target the biotin uptake system 48,49 .Interestingly, the very first paper on using biotin conjugates for drug delivery in mammalian cells was not for its ability to bind to the biotin uptake protein [50][51][52][53][54][55][56][57][58] .The very first mention of synthesising a biotin conjugate for taking advantage of the biotin uptake system in animals was reported in 2001 on the oral absorption properties of biotinylated retro-inverso Tat nonapeptides 59 .This study demonstrated an enhancement of up to 500-fold in transporting the biotinylated version of the peptide.Such transport was shown to be concentration dependent, saturable, and inhibitable by biotin.In 2004, a comparative study by Russell-Jones et al. 8 examined the uptake of fluorescently labelled polymers conjugated with biotin, folic acid, and vitamin B 12 in 18 different tumour cell lines in vitro and in vivo.Compared to the non-targeted polymer, an enhanced uptake of all the ligandconjugated polymers was found in various cell lines.For instance, Colo-26 cells (murine colon tumour) treated with vitamin B 12 -conjugated and biotin-conjugated polymers showed >2-fold more fluorescence intensity than that of non-targeted polymers.Of note, compared to the folic acid-conjugated and vitamin B 12 -conjugated polymers, the uptake of biotin-conjugated polymers was found to be >3-fold higher in M109 cells (murine lung carcinoma).Since then, biotin has been extensively studied as a vector for targeted delivery of anticancer agents 31,46,[60][61][62][63][64] .For information on biotin metabolism, biological functions, and its medical applications, readers are referred to several comprehensive reviews 31,32,[34][35][36]45,[65][66][67][68][69][70][71][72][73][74][75][76][77][78] .
While there has been very impressive progress in using biotin conjugates for targeted delivery applications in cell culture and animal models, much less is known about the mechanistic aspects of such success.Foremost, it is not clear whether such targeted delivery relies on a biotin transporter or receptor.Though literature discussions seem to use the terms biotin "receptor" and "transporter" without a clear distinction, these are indeed two distinctly different concepts 46,[79][80][81][82][83][84][85][86] .It is commonly accepted that "transporters mediate the passage of molecules across cell membranes by alternating between inward-and outward-facing states, while receptors undergo intracellular structural rearrangements that initiate signaling cascades 87 ."Known biotin transporters almost universally require the presence of a free carboxylic acid group for the recognition of biotin (Figure 1) 33,72,82,[88][89][90][91][92] , and yet the biotin conjugates used in targeted delivery transforms the carboxyl group to an amide or ester 32,45 , which is supposed to abolish the ability for a biotin transporter to recognise such a conjugate 72 .At the same time, no known "biotin receptor" has been identified, only transporters 72,73,88,91,93 .Further, there is a report of the "anti-inhibition" effects of biotin in the binding of a biotin conjugate with cells that express the biotin transporter 94 .In addition, a study by Wei and coworkers reported the lack of competition between 2000-fold excess of biotin and biotin conjugate during the uptake experiment in E. coli 95 .All these suggest the need for much more mechanistic studies on how biotin conjugation allows for "biotin-receptor/transporter"-mediated delivery.In this review, we provide a brief overview of the known biotin transporters, discuss known structure-activity relationships (SARs) in binding between a substrate and a biotin transporter, explore the disconnect between the SAR results and "biotin receptor/ transporter-mediated" delivery of biotin conjugate, and suggest various possibilities.All these are aimed to help future design of biotin conjugates for efficient delivery with a firm understanding of the biology behind it.Below, we provide our critical analysis by starting with a description of the various known biotin transport systems.

Known biotin uptake systems
The uptake of biotin into cells occurs mainly via the sodiumdependent multivitamin transporter (SMVT) 96 , which employs a transmembrane Na þ gradient for its translocation (Figure 2) 32 .In addition, a few studies have revealed a carrier system other than SMVT that might mediate biotin uptake in human peripheral blood mononuclear cells (PBMC) 97 and keratinocytes 98 .In the following subsections, we briefly discuss all the known biotin uptake systems in the mammalian cell.

Sodium-dependent multivitamin transporter
SMVT, a product of the solute carrier family-5 member-6 (SLC5A6) gene, is responsible for the transportation of biotin, lipoic acid, and pantothenic acid (Figure 1) across the cell membrane 70,72,99 .This transport is coupled with Na þ flux 99 .As proposed based on topographical analysis 100,101 , the SMVT consists of 635 amino acids and has 12 putative transmembrane domains with N-and C-termini towards the cytosol.The nucleotide sequence of the cDNA predicts a molecular weight of 68.6 kDa 70,102 .It also has four N-linked glycosylation sites in extracellular domains and two protein kinase C-dependent phosphorylation sites in cytoplasmic domains 100,103 .SMVT distributes ubiquitously in the human body and is most abundantly expressed in the absorptive tissues of the liver, intestine, placenta, pancreas, and kidney 100,104 .Functionally, SMVT transports solutes such as biotin across the cell membrane against an electrochemical gradient of Na þ .One biotin molecule is co-transported with two sodium ions in a single transport cycle (Figure 2) 100 .Again, SMVT has been widely studied for targeted delivery of biotin conjugates into cells overexpressing SMVT 105 ; although, known SAR binding for SMVT does not support such delivery.

Monocarboxylate transporter-1 (MCT-1)
The MCT family of proteins belongs to the proton-dependent transport protein family (SLC16A), with 14 members based on sequence homology 106,107 .These transporters mediate the uptake of short-chain monocarboxylates such as lactate, a-hydroxybutyrate, pyruvate, and biotin in mammalian cells 96 .Among these proton-dependent transporters, MCT-1 to MCT-4 mediate the uptake of endogenous monocarboxylates into the brain, kidney, and intestinal cells through the electroneutral co-transport of one monocarboxylate molecule with one proton (1:1) 108 .MCT-1 has been reported as an alternative biotin transporter in mammalian lymphoid cells; although, these cells also express SMVT 97 .MCT-1 utilises a sodium-dependent co-transport mechanism for uptake into the PBMCs 109 .The Michaelis-Menten constant (K m ) was reported to be 2.6 nM, �1000-times lower for MCT-1-mediated biotin transport in PMBCs than SMVT-mediated biotin transport 97,109 .Of note, carrier-mediated transport exhibits Michaelis-Menten kinetics; hence, K m is a widely used parameter to indicate the binding affinity (alternatively, specificity) of the substrates with the carrier proteins [110][111][112][113] .

Miscellaneous: uptake of biotin into keratinocytes
The liver, intestinal epithelia, kidney, and placenta express SMVT for biotin uptake 97 .In addition, Grafe et al. 98 studied the uptake of biotin in the human HaCaT cell line and native non-transformed keratinocytes and revealed that lipoic acid and pantothenic acid inhibit the Na þ -dependent biotin uptake in keratinocytes.SMVT showed an apparent Michaelis-Menten constant of 23 mM for biotin, 1 mM for pantothenic acid, and 4.6 mM for lipoic acid (oxidised form).In addition, kinetic studies showed the presence of a second uptake system for biotin in the human keratinocytes with an apparent Michaelis-Menten constant of 2.6 nM.Interestingly, this value is same as that for the MCT-1-mediated biotin transport in PMBCs.This uptake system seems specific for biotin because the uptake was not inhibited by lipoic acid and pantothenic acid.In comparison, SMVT-mediated biotin uptake was significantly affected by both lipoic acid and pantothenic acid.Hence, the study concluded that human keratinocytes express both the SMVT and another biotin-specific carrier-mediated transport system.

The structure-activity relationship: what might the SAR imply in terms of uptake mechanism(s)?
SMVT exhibits broad ligand specificity as it recognises a range of molecules such as biotin, pantothenic acid (vitamin B 5 ), a-lipoic acid, and iodide for their cellular uptake 31,70,114 .Another transporter MCT-1 recognises short-chain aliphatic acids such as lactate, pyruvate, butyrate, acetoacetate, b-hydroxybutyrate, and c-hydroxybutyrate and seems to be an even more versatile transporter than SMVT 96,106 .Because MCT-1 is largely involved in the transport of endogenous aliphatic carboxylates and has less prominent roles in biotin transport, it has not been as widely studied as the SMVT in terms of understanding the SAR for their binding characteristics.Therefore, we analyse the SAR of SMVT substrate/ligand characteristics as summarised in Table 1.The substrate specificity of SMVT has been studied by determining the uptake kinetics of radiolabeled biotin in the presence of unlabelled biotin and its structural analogues such as a-lipoic acid, a-lipoic amide (a-lipoamide), biocytin, desthiobiotin, biotin methyl ester, and pantothenic acid (Figure 1) 89,91,92,116 .For instance, Nylander and coworkers examined the uptake of biotin (5 mM) in the presence of its structural analogues in human intestinal brush border membrane (entry 1, Table 1) 91 .At 20 and 50 mM, desthiobiotin inhibited the uptake of biotin to 5.25 ± 0.18 pmol.mg protein −1 .20 s −1 and 4.88 ± 0.14 pmol.mg protein −1 .20 s −1 , respectively, when compared to the control group (9.80 ± 0.60 pmol.mg protein −1 .20 s −1 ).Similarly, a-lipoic acid also diminished the uptake to 5.60 ± 0.22 pmol.mg protein −1 .20 s −1 and 4.70 ± 0.27 pmol.mg protein −1 .20 s −1 at 20 and 50 mM concentrations, respectively in comparison to the control group.However, uptake studies using biotin analogues having modified carboxylic acid such as biotin methyl ester and a-lipoic amide did not result in a marked decrease in biotin uptake by intestinal cells.Another study done by Mitra and coworkers investigated the uptake of 10 nM [ 3 H]biotin in a human-derived retinoblastoma cell line (Y-79) (entry 4, Table 1) 93 .In the presence of 1 mM of unlabelled biotin, valeric acid, pantothenic acid, a-lipoic acid, and desthiobiotin, the uptake of [ 3 H]biotin decreased to 9.03 ± 1.51%, 60.39 ± 4.02%, 9.12 ± 1.39%, 6.97 ± 0.82%, and 15.61 ± 2.41% of the control group, respectively.Treatment with 1 mM of biocytin and biotin methyl ester did not produce any inhibitory effect on [ 3 H]biotin uptake.Therefore, it is clear that SMVT does not interact with biotin analogues having modified carboxyl group.Further, Said and coworkers conducted a similar study in the human intestinal cell line Caco-2 (entry 2, Table 1) 88 .Specifically, incubation of cells with [ 3 H]biotin (4 nM) with 25 mM of biotin and desthiobiotin resulted in the decrease of [ 3 H]biotin uptake from 1.69 ± 0.04 pmol.mg protein −1 .3min −1 to 0.664 ± 0.03 and 0.95 ± 0.02 pmol.mg protein −1 .3min −1 , respectively.However, no significant inhibitory effects on [ 3 H]biotin uptake were observed upon incubation with 25 mM of diaminobiotin, biotin methyl ester, and biocytin.A few conclusions can be drawn.First, desthiobiotin (devoid of the tetrahydrothiophene ring present in biotin) shows strong inhibition of biotin uptake by SMVT (entries 1-5) 33,88,89,91 .Similarly, a-lipoic acid (devoid of the tetrahydroimidazolone ring present in biotin) competes strongly with biotin for SMVT-mediated transport (entries 1, 3, and 4) 33,91,93 .Such results indicate that the presence of two fused five-membered rings is not essential.Second, diaminobiotin with two ionisable (protonation) amino groups on a single five-membered ring does not compete strongly with biotin (entry 2, Table 1).In addition, the 2-iminobiotin compound (Figure 1) that contains fused rings do not compete with biotin for SMVT-mediated transport.Obviously, 2-iminobiotin has a protonatable guanidine moiety instead of a urea moiety in the second five-membered ring.Though the SAR for biotin interaction with the SMVT seems to be complex 59,88,100 , the presence of a protonatable functional group clearly is not favourable for such interactions.Third, valeric acid was also found to be a strong inhibitor of biotin transport by the SMVT although it does not contain fused tetrahydroimidazolone and tetrahydrothiophene rings in its structure (entries 3 and 4, Table 1) 33,93 .The presence of a free aliphatic carboxylic acid group is the only common feature that exists among these structurally diverse substrates of SMVT117.As expected, modification of the carboxylic acid group to an ester (e.g.biotin methyl ester) or amide (e.g.biocytin and a-lipoic amide) led to the abolishment of the ability to compete with biotin for transport by SMVT (entries 1, 2, and 5, Table 1) 88,89,91,118 .
An extensive study by Finn and coworkers 119 demonstrates the high specificity of SMVT towards modification in the structure of pantothenic acid (Figure 1).In the study, pantothenic acid derivatives were tested for competitive reduction of [ 3 H]biotin uptake by SMVT in human embryonic kidney (HEK) cells transfected with human SMVT using LC-MS/MS.The SAR analysis (totally 18 derivatives) showed that modification of the carboxylic acid to an amide, ester, alcohol, or triazole of the b-alanine fragment of the pantothenic acid resulted in a significant loss of biotin uptake inhibitory activity.Further, it was found that only pantothenic acid derivatives with a free carboxyl group were tolerated by the transporter as shown by inhibition of biotin uptake and sodiumdependent transport of these derivatives.Overall, this study shows the high specificity of SMVT towards the modifications in the pantothenic acid structure and delineates the importance of the free carboxylic acid group of pantothenic acid for SMVT-mediated transport.Such results further corroborate the data from biotin uptake studies discussed in the previous SAR section, which emphasises the need for a free carboxylic acid group for SMVTmediated transport.
All the studies point to one thing: the presence of a carboxylic acid group is essential 72 .If one examines the mechanism through which SMVT transports a substrate, the requirement for a carboxylic acid is consistent with the driving force being electrochemical via a sodium ion gradient (Figure 2).In this regard, Table 1 and Figure 3 provide a summary of results from various competition experiments to show the structural properties of biotin crucial for its recognition by SMVT. .In such a case, the hydrolysed product would effectively compete with the [ 3 H]biotin for uptake by SMVT.
One of the central questions for this review is the vast body of literature examples of various drug-biotin conjugates synthesised through amidation or esterification of the biotin carboxylic acid group 8,31,45,120,121 .Despite the requirement of a free carboxyl group for SMVT-mediated transport and the lack of a carboxyl group in commonly used biotin-drug conjugates, most studies attribute the targeted delivery of such conjugates due to binding to a "biotin receptor," which is sometimes inaccurately used to mean biotin transporter.As discussed earlier, the concept of a "receptor" is different from that of a "transporter".In the subsequent sections, we provide a brief description of such examples and analyse the apparent inconsistency between the structural need for SMVT transport and the key features of such biotin conjugates.

Biotin conjugates in delivery applications
Biotin has been extensively investigated for various applications including delivery of both therapeutic and imaging agents 31,45,69,78,86,122 .Without being comprehensive, we herein attempt to succinctly show the extent of applications of biotin conjugates.Table 2 displays examples of various types of applications of biotin conjugates.Mostly, biotin has been used as a vector to deliver a payload to cancer cells 31,86 .For instance, Kim and coworkers published their work on the development of a biotinconjugated theranostic agent (compound 2) containing gemcitabine (an anticancer drug) and coumarin (a fluorescent reporter) (entry 2, Table 2) 123 .As proposed by the authors, this multifunctional compound undergoes thiol-triggered release of gemcitabine and biotin-conjugated coumarin under physiological conditions.Briefly, compound 2 (10 mM) was incubated for 15 min with A549 cells (human lung carcinoma epithelial cells) and WI38 cells (Caucasian fibroblast-like foetal lung cells).Markedly, higher fluorescence was observed in A549 cells compared to the WI38 cells.Further, compound 2 was tested for anticancer activity using a cell viability assay.In comparison to the control group (no treatment), compound 2 reduced the cell viability by >80% at 1 mM, while non-biotinylated gemcitabine-coumarin conjugate reduced the cell viability by �50%.In addition, gemcitabine alone (1 mM) treatment resulted in �30% reduction of the cell viability compared to the control group (untreated).Subsequently, Kim and coworkers also reported biotin-coumarin conjugate (compound 1) for selective labelling of cancer cells (entry 1, Table 2) 121 .In this study, biotin-conjugated coumarin (5 mM) showed a high level (quantitative data not provided) of fluorescence in A549 cells compared to WI38 cells after 20 min of incubation.Additional cell imaging studies showed a relatively high level of fluorescence (quantitative data not provided) in A549 cells for the biotin-conjugated compound when compared to its non-biotinylated derivative after 20 min of incubation.Overall, results from both articles show the ability of biotin to selectively deliver compounds to cancer cells.Though the enhancement effects seem small, biotinylation of anticancer drugs indeed provides beneficial effects.Additionally, biotin conjugation has been also extensively exploited for enhancing cancer cell accumulation of anticancer compounds such as SN-38 (entry 3, Table 2), SB-T-1214 (entry 4, Table 2), cisplatin (entry 5, Table 2), curcumin (entry 8, Table 2), squamocin (entry 9, Table 2), and paclitaxel (entry 10, Table 2) 46,80,124,[126][127][128] .Generally, such biotin conjugates are synthesised through amidation or esterification of the biotin carboxylic acid (Figure 4).As discussed in the previous section, such transformations are contraindicated for interaction with the SMVT based on SAR studies and beg the question as to whether transport of such biotin conjugates is through the SMVT.In the subsequent sections, we probe the consequences of modification of the carboxylic acid on the transport of biotin conjugates reported in the literature for various applications.

Activated biotin for cell-surface protein labelling work
Relevant to the discussion of biotin-mediated drug delivery issues, it is important to address another seemingly contradictory application of biotin: selective covalent labelling of (only) cell-surface proteins using biotinylated reactive agents [129][130][131][132][133][134][135][136] .Such experiments utilise the high acylating reactivity of N-hydroxysuccinimide-biotin conjugates (NHS-biotin) and sulfo-NHS-SS-biotin (Figure 1) to specifically conjugate and subsequently extract cell-surface proteins using streptavidin beads 130,131,134,137 .Such a design is predicated on two characteristics of biotin: low passive permeability across the cell membrane (and thus only labels cell surface proteins) and high affinity for streptavidin (and avidin) 122 , which are bacterial proteins with high affinity for biotin and are often used in isolation of biotinylated conjugates.Furthermore, such protein labelling work is not limited to cells without biotin transporter(s).A natural question is whether such protein labelling experiments would contradict the idea of transporter-mediated uptake of biotin conjugates.The answer seems to be with the temperaturedependent nature of the biotin transport 33,46,79,89,93 .Generally, cell-surface labelling experimental protocols apply low temperature (generally performed at 4 � C) during the incubation of biotinylating reagents with cells to minimise uptake and afford selectivity for surface proteins 129,133,138 .However, there are also examples of labelling experiments conducted at room temperature or 37 � C [139][140][141][142] .It is not clear whether the success of such experiments is correlated with low or lack of expression of the biotin uptake system.At this point, this issue needs to be examined to understand the mechanistic implications.It is possible that the acylating reaction is kinetically faster than transport, leading to the selective labelling of cell surface proteins.Another possibility is for NHS-biotin to acylate any biotin transporter present, leading to covalent inhibition.All these also suggest the possibility of using NHS-biotin or similarly activated biotin-based acylating agent for identifying any unknown "biotin receptor".

Intriguing possibilities
With all the reports of targeted drug delivery by using biotin conjugates, there are very important unresolved issues.First, most of  In a cell viability assay, compared to the untreated group, biotinconjugated gemcitabine (1 mM) reduced the cell viability to <20% while the cell viability was found to be slightly >70% when treated with gemcitabine (1 mM) alone for 72 h. the SAR studies in biotin transporter binding uniformly specify the need for a carboxyl group.This is described in detail in section "The structure-activity relationship: what might the SAR imply in terms of uptake mechanism(s)?"(Figure 3 and Table 1).However, with all the biotin conjugates, the carboxyl group is used for conjugation through the formation of an amide (or occasionally an ester) bond.This means the elimination of the free carboxyl group.Then, there is the question as to why a conjugate with a modified carboxyl group binds to a transporter that has the structural requirement of a free carboxyl group in its substrate.Do these biotin conjugates really rely on the biotin transporter(s) for targeting and delivery of a payload?If not, what is/are the molecular target(s) for these biotin conjugates?Second, in the literature, the term "biotin receptor" is often used 79-81,83,85,86 .However, there has not been a "biotin receptor" reported 72,73,75,77,91,92,109,118 .Here, there is the need to emphasise that "transporter" and "receptor" are two different concepts in biology.Again, structurally "transporters mediate the passage of molecules across cell membranes by alternating between inwardand outward-facing states, while receptors undergo intracellular structural rearrangements that initiate signaling cascades 87 ".There will be a need to conduct extensive studies of this issue.If biotinbased drug delivery does not rely on a biotin transporter (or specifically SMVT), then why is the reported correlation of biotin transporter expression level with the ability for biotin conjugationbased targeting?Third, the biotin transporters tend to have K m in the low to mid-micromolar range 88,90,92,102,143 .Then why some biotin conjugates in the nanomolar range are effective in targeted delivery?Lastly, SMVT has a molecular weight of 68.6 kDa and transports small molecules such as biotin, a-lipoic acid, pantothenic acid, and valproic acid effectively 70,102 .However, there have been reports of biotin-mediated transport of payloads of high molecular weight and/or size such as biotin-conjugated polypeptides, synthetic polymers, and nanoparticles 31,84,103,144 .
Molecular size plays a vital role in determining its cellular uptake mechanism.For example, nanoparticles tend to undergo an endocytic uptake mechanism due to their large size while small molecules may enter cells passively or via active transport depending on their physiochemical properties [145][146][147] .In the following subsections, we pose these questions and present our analysis.

Transporter vs. receptor
Again, earlier SAR studies have uniformly shown the critical role of the carboxylic acid group for SMVT-mediated uptake of biotin.However, biotin conjugates in targeted delivery studies uniformly lack this functional group.For instance, Chen et al. 103 developed biotinylated chitosan surface-modified poly(D,L-lactide-co-glycolide) nanoparticles (Bio-PLGA NPs) to deliver epirubicin (anticancer agent) to MCF-7 cells in vitro.Compared to epirubicin-loaded PLGA (54.5%), cell viability was somewhat lower (36.1%) when treated with Bio-PLGA NPs for 120 h.The cytotoxic effect exerted by Bio-PLGA NPs was attributed to the enhanced accumulation of the drug inside cancer cells.Cellular internalisation of NPs was investigated by measuring epirubicin fluorescence signals using flow cytometer.In MCF-7 cells, a �41% increase in fluorescence was observed after treatment with Bio-PLGA NPs for 2 h in comparison to epirubicin-loaded PLGA.Furthermore, experiments were carried out to understand the mechanism of the cellular accumulation of Bio-PLGA NPs.In this regard, cells were pre-treated with 10 mg/mL (40.93 mM) of biotin in serum-free media at 37 � C.After 24 h, cells were washed and treated with Bio-PLGA NPs at epirubicin concentration of 10 mg/mL (18.4 mM) for 2 h and further analysed by flow cytometry.Pre-treatment with biotin reduced the uptake of Bio-PLGA NPs to �60% of the control group.On the surface, the results seem to be consistent with biotin transportermediated accumulation.Further, endocytic inhibitors chlorpromazine, filipin, and amiloride were used to probe the mechanism of  2.
receptor-mediated endocytosis.In all the endocytosis experiments, endocytic inhibitors were incubated with MCF-7 cells for 24 h at 37 � C prior to the incubation with NPs for 2 h before flow cytometry analysis.Chlorpromazine is commonly used to inhibit clathrinmediated endocytosis, which is widely regarded as a major pathway to transport cargo through endocytosis [148][149][150][151] .Amiloride acts as an inhibitor of micropinocytosis by inhibiting Na þ /H þ exchange proteins on the cell membrane 152 .In addition, caveolae-mediated endocytosis can be inhibited by filipin 153 .These pathways have been classified as fundamental routes for intracellular trafficking of various extracellular molecules including nutrients, hormones, and nanoparticle 149,154,155 .By measuring epirubicin fluorescence using flow cytometry, a >50% reduction of cellular internalisation of Bio-PLGA NPs was noted after pre-treatment with chlorpromazine (10 mg/mL) compared to the control group.About 15% and 30% reduction of Bio-PLGA NPs cellular uptake were noted after pre-treatment with filipin (5 mg/mL) and amiloride (10 mg/mL), respectively, when compared to the respective control groups.
The inhibitory effect of amiloride on biotin transport is well-known as the blockade of the Na þ channel, suggesting a possibility of impairment of sodium-dependent transport of biotin by SMVT 117 .Interestingly, results also suggest a receptor-mediated process, since SMVT transporter does not require endocytosis for the translocation of biotin into the cells.Endocytosis is part of the receptor-mediated trafficking of cargo 156,157 .However, the identity of this "receptor" is not known and is probably not the biotin transporter.In contrast, a study by Mitra and coworkers reported that the intracellular uptake of [ 3 H]biotin remains unaltered in humanderived prostate cancer cells (PC-3) after pre-treatment with colchicine (100 mM) compared to the control group 117 .Noteworthy, colchicine inhibits endocytosis by disrupting microtubule assembly 109 .Sinko and coworkers 158 also presented evidence against endocytosis 103 being the uptake mechanism.Briefly, enhanced accumulation of biotinylated polyethylene glycol (PEG)-based Tat9 peptide (PEG:(R.I-Cys-K(biotin)-Tat9) 8 ) (29 kDa) was observed in Chinese hamster ovary (CHO) cells transfected with human SMVT (CHO/hSMVT).In the competition experiments, biotin-conjugated peptide (0.1 mM) was co-incubated with 50 mM of various compounds.After 10 min coincubation, free biotin diminished the uptake of biotin-conjugated peptide to �35%; desthiobiotin and biocytin inhibited the uptake to �45% as compared to the control group.Further, pantothenate, PEG-biotin (PEG MW ¼ 3400), and biotin-PEG-biotin (PEG polymer containing two biotin molecules) reduced the uptake of the biotin-conjugated peptide to �55-60% of the control group.Such results show competitive inhibition of biotin-conjugated peptides by molecules with a free carboxyl group (biotin, desthiobiotin, and pantothenate) as well as molecules with an amide group (biocytin, PEG-biotin, and PEG-biotinbiotin).Overall, results from these studies further emphasise the need to carefully look into the molecular mechanism(s) of biotin transport.Along this line, Russell-Jones et al. published a comprehensive study investigating the participation of an SMVT-independent uptake system in the absorption of biotin-conjugated rhodamine-labelled hydroxypropyl-methacrylamide (Bio-Rh-HPMA; 22 kDa) and biotin-conjugated quantum dots (B-Qdots; 10-12 nm) 116 .Bio-Rh-HPMA was synthesised through conjugation using biotin's carboxylic group.Further, different tumour cells such as ID8, RENCA, MMT, and Ov2008 were chosen to study the in vitro uptake mechanism of Bio-Rh-HPMA (25 mL of 20 mg/mL) using fluorescent microscopy.After 4 h incubation, histological examination showed punctate staining of the tumour cells due to the accumulation of the fluorescent Bio-Rh-HPMA in vesicle-like structures, implying endosomal uptake of the Bio-Rh-HPMA.
Further, experiments were also carried out in vitro with B-Qdots.RD995 cells were incubated with 2.5 mL B-Qdots in 500 mL culture medium and monitored at 1 h and 5 h time points.The concentration B-Qdots was not provided in the article.However according to the catalogue (catalogue number: Q10321MP) from the manufacturer (Invitrogen, Carlsbad, CA), it should be 2 mM (stock solution).Hence, cells were incubated with 10 nM B-Qdots.A time course experiment using a fluorescent microscope showed clustering of the fluorescent B-Qdots on the cell surface at 1 h, with minimal internalisation.B-Qdots in subcellular vesicles were observed after 5 h.The phenomenon of surface-clustering and endosomal uptake are the characteristics of receptor-mediated uptake of cargo 156,157,[159][160][161] .Such results are not strongly consistent with polymer uptake by SMVT.Furthermore, the uptake of Bio-Rh-HPMA (100 mL of 20 mg/mL for 6 h) in M109 metastatic tumourbearing mice was also studied.Imaging studies showed that Bio-Rh-HPMA was predominantly absorbed by the tumour cells growing immediately adjacent to normal intestinal tissue.As normal intestinal cells also express SMVT 32 , the selective uptake of fluorescent molecules by tumour cells suggests the possibility of an SMVT-independent uptake system responsible for the absorption of Bio-Rh-HPMA in tumour cells.Clearly, analysis of these studies raises several questions that warrant further investigations to delineate the cellular uptake mechanism of biotin and its conjugates.
Adding to the perplexity, a study by Smirnov and coworkers 94 revealed unique results in a competition assay between biotin and biotin-conjugate with PEG through an amide bond.In this study, attachment of HeLa cells onto the flat glass surface was examined.The glass surface was decorated with 1% biotin through a PEG linker.For the competition assay, the cell suspension was mixed with 0.8 mM biotin.Within 1 min, the cells were further incubated for 15 min with modified glass slides to probe the attachment of the cells on the surface.Unlike the reduction of biotin-conjugate uptake in the presence of biotin as reported by Chen et al. 103 and other researchers 46,63,162-164 , a 3-4-fold enhancement in attachment of cells onto the modified glass surface was recorded when compared to experiments without added biotin.Similarly, an augmentation of attachment of cells onto the modified glass surface was also observed with MCF-7 cells in the competition experiment.Incidentally, a study by Wei and coworkers reported that excess biotin (4 mM) did not impair the uptake of biotinylated Atto565 (2 mM) in a competition assay in E. coli 95 .In this study, the authors investigated the impact of biotin conjugation on the accumulation of Atto565 (a commercially available fluorescent dye) in E. coli.Interestingly, fluorescence measurement of the biotin-conjugated Atto565 showed �2-fold higher accumulation of Atto565 upon biotinylation.Once again, such unexpected results highlight the differential mechanism involved in the interaction of biotin and biotin-derivatised molecules with their specific uptake system(s).Given the wide-spread interest in using biotin conjugates in targeted drug delivery, these results suggest the need for further mechanistic studies to probe the cellular uptake of biotin conjugates.

The Michaelis-Menten constant
Similar to enzymes, SMVT also displays Michaelis-Menten saturation kinetics to transfer substrates across the cell membrane 33,113,158 .Using this Michaelis-Menten equation, several researchers have extensively studied SMVT-mediated uptake of biotin.Previously, the K m value of biotin was calculated to be 9.5 mM in Caco-2 cells 88 , 32.52 mM in rabbit corneal epithelial cells, 63.8 mM in Statens SerumInstitut Rabbit Cornea (SIRC) cells 90 , and 19.7 mM in human colonic epithelial NCM460 cells 92 .Along this direction, Sinko and coworkers have investigated the uptake of a biotin conjugate, i.e. [ 3 H]PEG-biotin (PEG MW ¼ 3400) in Caco-2 cells 158 .The kinetic studies showed significant inhibition of [ 3 H]PEG-biotin uptake by free biotin with an inhibition constant (K i ) of 6.78 mM.The K i for desthiobiotin, biocytin, and biotin-PEGbiotin was calculated to be 11.47, 14.01, and 19.08 mM, respectively.The K m value of biotin-PEG was calculated to be 6.61 mM in Caco-2 cells.Once again, these results clearly show competitive inhibition of biotin-PEG by biotin and its derivatives without a free carboxylic acid.Noteworthy, biocytin has been reported to be a weak competitor of biotin transport by SMVT in T47D cells (entry 5, Table 1) 89 , Caco-2 88 , and other cell lines 89,118 .The K m value of biotin-PEG was also found to be similar as shown by biotin towards SMVT in Caco-2 cells 88 .Based on the K m values, authors stated that both biotin and its conjugates devoid of a carboxylic acid group share the same uptake mechanism.Simultaneously, such results also contradict earlier findings, which indicate the essential nature of a free carboxylic acid group for SMVT-mediated transport.Hence, this example further emphasises the need to carefully examine the molecular mechanism(s) of biotin transport.

Small vs. large molecule
In the past two decades, biotin has been widely used as a targeting ligand for delivering small molecules such as doxorubicin, gemcitabine, and large molecules such as polypeptides, polysaccharides, nanoparticles, and micelles 31,45,116,162,165 .If we take a look at the molecular weights of the currently known SMVT substrates, all of these molecules are small molecules (Figure 1).Of note, the molecular weight of SMVT is calculated to be 68.6 kDa 70,102 .Generally, molecules <1 kDa molecular weight are categorised as small molecules 145,147 .However, it is imperative to ask the question of whether biotin-conjugated small molecules share the same uptake mechanism as conjugated macromolecules or particles.For instance, Chen et al. applied Bio-PLGA NPs (10-30 kDa) to selectively deliver epirubicin to cancer cells 103 .These nanoparticles have 50-150 times higher molecular weights than biotin.Generally speaking, there is the question of whether a transporter meant for a small molecule is capable of transporting a nanoparticle.As described in the previous section, Bio-PLGA NPs undergo the endocytosis pathway to enter cells as demonstrated by the experiments applying endocytosis inhibitors, viz., chlorpromazine, filipin, and amiloride.Similarly, experiments conducted by Russell-Jones et al. employing Bio-Rh-HPMA (22 kDa) also indicate the participation of endocytic pathways to deliver Bio-Rh-HPMA to tumour cells as demonstrated by imaging studies 116 .Therefore, these findings point to the possibility of the presence of multiple mechanisms for the uptake of such biotin-conjugates that are devoid of a carboxylate group and comprise high molecular weight.Endocytosis for the trafficking of biotinylated cargos seems to be a distinct possibility as shown by the experiments applying endocytosis inhibitors.

Conclusions
Biotin has been widely studied as a vector for targeted drug delivery.SMVT has been recognised as the major transporter of biotin.Based on SAR studies, it has been found that the free aliphatic carboxylic acid of biotin plays a vital role in its recognition by SMVT.Importantly, the modification of the carboxylic acid of biotin to an amide or ester functional group seems to abolish its SMVT-mediated uptake.However, biotin-mediated drug delivery relies on the payload moiety being conjugated through amidation or esterification of the free carboxylic acid of biotin.Despite being devoid of free carboxylic acid, these biotin conjugates are reported to target biotin uptake system(s) for enhanced cell permeation.The results from the competition assay of biotin conjugates with free biotin and endocytosis experiments imply the involvement of multiple mechanisms for the uptake of biotin conjugates.In this regard, further studies are warranted to delineate the cellular uptake mechanism of biotin conjugates.A clear understanding of such mechanism(s) will be critical for the future design and development of biotin-based therapeutics.

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

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

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

Figure 4 .
Figure 4.Chemical structures of the biotin-conjugates described in Table2.

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

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