key: cord-0971209-lup5q9wk
authors: Dałek, Paulina; Drabik, Dominik; Wołczańska, Halina; Foryś, Aleksander; Jagas, Małgorzata; Jędruchniewicz, Natalia; Przybyło, Magdalena; Witkiewicz, Wojciech; Langner, Marek
title: Bioavailability by design – Vitamin D(3) liposomal delivery vehicles
date: 2022-03-26
journal: Nanomedicine
DOI: 10.1016/j.nano.2022.102552
sha: d8221493b187bbd5a8a873dbdb9683c9aab1e1cf
doc_id: 971209
cord_uid: lup5q9wk

Vitamin D3 deficiency has serious health consequences, as demonstrated by its effect on severity and recovery after COVID-19 infection. Because of high hydrophobicity, its absorption and subsequent redistribution throughout the body are inherently dependent on the accompanying lipids and/or proteins. The effective oral vitamin D(3) formulation should ensure penetration of the mucus layer followed by internalization by competent cells. Isothermal titration calorimetry and computer simulations show that vitamin D(3) molecules cannot leave the hydrophobic environment, indicating that their absorption is predominantly driven by the digestion of the delivery vehicle. In the clinical experiment, liposomal vitamin D(3) was compared to the oily formulation. The results obtained show that liposomal vitamin D(3) causes a rapid increase in the plasma concentration of calcidiol. No such effect was observed when the oily formulation was used. The effect was especially pronounced for people with severe vitamin D(3) deficiency.

In classical pharmacology, the oral bioavailability of an active compound depends predominantly on its solubility in the aqueous phase and the passive transfer across structural barriers such as the epithelium or the Blood-Brain Barrier. Such properties impose specific structural requirements on an active compound as formulated by the rule of five, first published by Lipinski et al. [1] . The approach is applicable only for active compounds that are moderately hydrophobic (logP < 5). A compound hydrophobicity is typically quantitated by the log of the octanol/water partition coefficient (logP) [2] [3] . However, for very hydrophobic molecules, as exemplified by cholesterol and its metabolite vitamin D 3 (cholecalciferol), the classical approach cannot be applied [4] [5] . Highly hydrophobic compounds cannot enter inner body compartments unassisted. This is well illustrated by the absorption and biodistribution of cholesterol, which with logP > 7 requires complex supramolecular carriers in the form of lipoproteins, dedicated receptors, and complex redistribution mechanisms within the cell [6] [7]. Vitamin D 3 is an essential hydrophobic compound necessary for a variety of metabolic and immunological processes. Its importance is demonstrated by the fact that it controls more than 700 genes, and its deficiency can lead to a number of pathologies [8] [9] . For example, as recently demonstrated, vitamin D 3 supplementation reduces the severity of the disease caused by COVID-19 infection [10] [11] [12] . Therefore, its effective delivery is a prerequisite for effective supplementation. Vitamin D 3 can be acquired by humans from both intrinsic and extrinsic sources. The intrinsic source requires exposure to organism. To design an effective delivery strategy, the absorption mechanisms of hydrophobic compounds from the diet need to be considered in detail [16] . For subsequent analysis, vitamin D 3 absorption and distribution processes can be functionally separated, as its absorption is mainly dependent on specialized epithelial cells in the gut, whereas the fluxes of vitamin D 3 and its metabolites inside the body are tightly controlled by dedicated transport mechanisms and metabolic processes [17] . Transfer of vitamin D 3 and its metabolites within the body cannot be altered by simple adjustments in supplement formulation and/or dose. The digestion and absorption processes, on the other hand, can be greatly altered by modifications of the vitamin formulation [18] . Water-insoluble hydrophobic compounds require complex enzymatic and/or physicochemical mechanisms to facilitate their internalization [19] .

Typically, highly hydrophobic compounds remain during the digestion process within supramolecular aggregates [20] [21]. The digestion and subsequent absorption of particulates with the hydrophobic compound are restricted by mucus, which contains a complex polymer matrix made of mucins [22] . Consequently, epithelial cells are only accessible to particulates smaller than 300 -500 nm [23] . In addition, the absorption of a particulate carrier can be facilitated only by competent cells [24] [25] . These cells can absorb intact particulates of certain sizes, therefore providing means for highly hydrophobic compound internalization.

Liposomal formulation of vitamin D 3 was prepared by mixing two solvents: propylene glycol containing lipids (20% w/w in the final preparation) with vitamin D 3 and purified water (1:1 w/w) followed by extrusion through the 100 nm polycarbonate filter. The high content of lipids in the mixture results in high viscosity and gel-like consistency of the suspension.

Finally, the lipid gel was diluted 200 times with the glycerol/water (1:1 w/w) mixture and supplemented with natural flavor (0.3 w/w) and pectin (2.45 w/w). The quantity of propylene glycol (E490) in a single serving (5g) of the final liposomal formulation is three orders of magnitude lower than the official limits considered as safe for oral formulations ( [26] and references therein). After each preparation of the liposome suspension, the size and polydispersity index were measured to confirm the formation of the monodisperse population of liposomes. Due to its high hydrophobicity (logP > 7), it can be assumed that all cholecalciferol is located in the lipid bilayer forming the liposome wall ( [27] and the citations therein); therefore, its encapsulation efficiency is expected to be high. For control studies, the commercially available oily solution of vitamin D 3 was used. In this case, the quantity of vitamin D 3 was assumed to be equal to the value declared by the producer.

The size distribution of liposomes in the liposomal vitamin D 3 formulation was determined by the dynamic light scattering method with some modifications in the preparation of the measured samples due to the presence of pectin. Liposomes were extracted from the liposomal formulation by diluting it 100 times in purified water followed by adding 0.1 sample volume of 1 M NaOH before measurement. The effect of the procedure on the integrity of liposomes was measured in the control experiment and no alteration of the liposome parameters (average size and polydispersity (PDI)) was observed (data not shown).

The quantity of vitamin D 3 was determined by RP-HPLC (Reversed-Phase High Liquid Chromatography) according to the method developed by Sazali et al. [28] with some modifications in the preparation of the sample. The details of the HPLC measurements are presented in the Supplementary Materials. The vitamin D 3 concentration used in the liposome formulation was two orders of magnitude lower than that with demonstrated toxic effects on cells in culture [29] .

The encapsulation efficiency (EE%) of vitamin D 3 in liposomes was evaluated using the ultrafiltration method combined with quantification of vitamin D 3 before and after extrusion.

The suspension of liposomes with vitamin D 3 was diluted 50 times with purified water and placed into Amicon Ultra Centrifugal Filters (volume 0.5 ml, NMWL 50k) to separate liposomes with vitamin D 3 and free vitamin D 3 . Samples were centrifuged for 15 minutes (14000 RPM). Then, the solution of free vitamin D 3 (permeate) were analyzed with the RP-HPLC method described by Sazali et al. [28] . Before and after ultrafiltration the size distribution of liposomes in the sample was determined to control their stability. The encapsulation efficiency of vitamin D 3 was calculated according to the following equation:

where m free is the mass of vitamin D 3 in the permeate and m total is the total mass of vitamin D 3 in the sample. Since vitamin D 3 is highly hydrophobic, when it reaches the saturation level in the lipid bilayer, it will precipitate in the aqueous phase. Consequently, to quantitate the free vitamin D 3 , the monomer and precipitate forms should be accounted for. To do this, quantities All samples were prepared in 20 mM HEPES buffer (pH 7.4). Before each experiment, their pH was measured and adjusted if necessary. The pH difference between the solutions did not exceed the 0.02 value, so the protonation effect did not interfere with the measurement.

Finally, the solutions were degassed shortly before measurements. In all experiments, the rotor speed was set at 250 rpm, the injection volume at 10 l and the time of single injection to 300 s (control measurements) or 500 s (measurement in the presence of vitamin D 3 , to detect the occurrence of any slow processes).

All full-atomic simulations were performed with the NAMD [32] software and united-atom CHARMM36 force field under NPT conditions for lipid molecules. Vitamin D 3 and Soy Triglyceride (STri) force fields were created using Scigress and parameterized based on quantum calculation (optimization and single-point calculation) [33] . Four different molecular systems were simulated: a single vitamin D 3 molecule with POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine) bilayer, 10 vitamin D 3 molecules with POPC membrane, STri system, and STri system with 10 VTD molecules. All systems were hydrated with TIP3P The size of the simulation box was 45x40x45 Å. The total simulation time was 50 ns.

Membrane thickness (d P-P ) was determined from the difference between the coordinates of phosphorus atoms in the opposite monolayers along the membrane normal. The surface area per lipid molecule in the membrane (APL) were determined using Voronoi tessellation.

Specifically, the membrane surface was divided into groups of n points (each point represented phosphorus and was labeled as P(x,y)) in such a way that each point was equally distant to neighboring n points. To exclude the possibility of overestimation, each membrane was multiplied in all three dimensions. Only parameters from the initial area were used for APL estimation. Every phosphorus at the distance from vitamin D 3 molecule smaller than 8 Å was considered as "perturbed".

The clinical experiment has been performed on healthy volunteers (age 24 -65) according to "the cross-over design". Volunteers were randomly divided into two groups. The general scheme of the clinical experiment is presented in Figure 1 . 

As stated above, liposomes designed for rapid and effective delivery of vitamin D 3 should have sizes smaller than 300 nm. Figure 2 shows size distribution of liposomes contaning vitamin D 3 . The fitting of the experimental data shows that the average liposome size and PDI are equal to 117 nm and 0.23, respectively. In addition, the suspension of liposomes prepared according to the procedure described above is stable, both from a microbiological and physicochemical point of view, for more than six months (Table S1 (Table S2 ). All of this shows that the encapsulation efficiency of vitamin D3 in liposomes in [47] . Figure   3A illustrates the hypothetical pathway of cholecalciferol internalization into the lipid bilayer. This indicates that vitamin D 3 spontaneous transfer across the lipid bilayer is unlikely due to the massive conformation changes required in both membrane monolayers [48] [49] .

Interestingly, when the cholecalciferol incorporation into the DOPC lipid bilayer is completed, the presence of the cholecalciferol molecule does not alter the lipid organization, as evaluated by the bilayer thickness and area per lipid molecule (two parameters frequently used to evaluate the perturbation caused by exogenous molecules [50] ). All this leads to the conclusion that cholecalciferol when in the lipid bilayer, is not likely to change its location due to the prohibitive energetic barriers required for movement along the membrane normal.

The behavior of vitamin D 3 is similar to that of cholesterol [51] .

In another simulation, several cholecalciferol molecules were positioned in the aqueous phase adjacent to the lipid bilayer, as shown in the first image in the emulsion towards reduced-size particulates will be greatly affected by the dietary fats from a foodstuff, which would reduce the ratio of bile acids and triglycerides, therefore, altering (slowing) the emulsification process. In addition, increased volume of substrate will also slow the digestion by lipases. When cholecalciferol is delivered in liposomes, with a size enabling the rapid mucus penetration followed by absorption assisted by proteins or endocytosis in enterocytes. Consequently, vitamin D 3 will rapidly enter the lymphatic system, followed by the metabolic transformation in the liver [52] [53] . The presented molecular arguments lead to the conclusion that vitamin D 3 delivered in liposomes will result in the appearance of calcidiol in the blood circulation much earlier than when delivered in the oily formulation.

The prediction that the liposomal formulation of vitamin D 3 will be absorbed in the gastrointestinal tract much faster than the oily formulation was tested in the medical experiment. In the experiment, vitamin D 3 was applied orally using two formulations; oilcontaining capsules and liposomes with sizes satisfying physiological requirements, i.e. their diameters are smaller than 300 nm. Size restriction reflects the structure of mucins in mucus and anticipated endocytic processes in enterocytes [54] [55] [22] [56] [57] . The hypothetical sequence of events for each formulation can be proposed, and the outcome of the experiment predicted. Specifically, triglycerides with cholecalciferol after swallowing will form an illdefined emulsion. Before entering the intestine, where the fat absorption is taking place, it will interact with foodstuff in the stomach. The inherently unstable oily emulsion can phase separate in the stomach what would increase the time of release to the intestine [58] . In addition, the phase separation will result in the reduced time of exposure of the oily emulsion to enterocytes due to its unequal distribution in the foodstuff. In the intestine, the oily emulsion will be digested and emulsified by the joint action of bile acids and lipases. Due to digestion processes, the emulsion will slowly evolve into a suspension consisting of oil J o u r n a l P r e -p r o o f Journal Pre-proof droplets and micelles, both containing vitamin D3. The digestion process will be greatly affected by the foodstuff, which will delay absorption. The ill-defined size of the oily solution and a tendency to coalescence will likely produce a wide range of heterogeneous particles that reduce their effective transport across mucus [57] . Both transendocytosis and receptormediated intake of vitamin D 3 require close particulates proximity to the intestine wall [59] [60] . Eventually, due to bile acids and the activity of enzymes, a fraction of the oily solution will be reduced to particulates smaller than 300 nm, including micelles capable to pass the mucus barrier and making vitamin D 3 available for internalization [57] [53] . The inherent uncertainty of the process and the limited time available for digestion will result in the outcome where only a fraction of vitamin D 3 will be absorbed. Suspension of uniform liposomes containing vitamin D 3 with sizes smaller than 300 nm, on the other hand, are predicted to behave differently. Firstly, the inherent stability of liposome suspension will mix well with the aqueous phase and will be little affected by foodstuff in the stomach.

Consequently, liposomes will spread uniformly throughout the whole volume of the stomach content, enhancing the effective exposure time of liposomes for absorption in the intestine.

Liposomes are not easily destabilized by low pH or mechanical stress and their topology is preserved even during enzymatic digestion [61] [31] . Only a sufficiently high quantity of surface-active substances such as bile salts, which will appear in the intestines, will destabilize the lipid bilayer. The action of detergents will result in much smaller mixed micelle formation, therefore, speeding up the vitamin D 3 transfer across the mucus layer even more [62] [63] . Consequently, it is expected that cholecalciferol absorption in liposomes will be superior to the oily solution with respect to the onset time of absorption.

To test this assumption, a cross-over clinical experiment has been performed. Healthy Continuous efforts are made to evaluate the bioavailability and pharmacokinetics of active substances as early as possible during the drug development process [65] . The introduction of targeted drug delivery systems opens new possibilities to modify the absorption and/or biodistribution of active substances without the need for its chemical modifications [19] [66] [67] . The effectiveness of the approach is elegantly demonstrated by the example of DOXIL TM . In this case, the structural features of the formulation are designed to achieve passive targeting, based on the structural properties of solid tumors and interaction with proteins in the circulation (long circulation time) [68] . Since then, a variety of delivery systems have been developed to improve an active substance solubility, enhance its affinity to target cells, reduce metabolic elimination, or achieve accumulation in specific locations within the body [69] [70] [71] . As shown in the paper, a similar approach can be employed to enhance the bioavailability of the orally delivered compounds which are not considered as drugs [4] [36] . The spatial and topological limitations imposed by the digestive processes can be implemented in the design of a delivery vehicle for highly hydrophobic compounds to improve its performance in vivo.

When designing an effective oral vitamin D 3 formulation, two qualitatively different processes need to be considered, absorption and redistribution. While absorption is greatly affected by vitamin D 3 formulation, redistribution is not. After internalization, transport and metabolic processes determine the quantity of vitamin D 3 and its metabolites redistributed [25] ). Consequently, cholecalciferol will remain in the nonpolar solvent during the entire digestion process or will precipitate. Therefore, its absorption will depend mainly on the properties of particulates and their fate during the digestion process [19] [23] [52] .

The hydrophobic compound such as vitamin D 3 can be delivered in three forms: as crystals, oily solution or preformed particulates in the aqueous suspension. Its low solubility in an aqueous phase makes crystal form impractical since its absorption is expected to be negligible [25] . When hydrophobic Vitamin D 3 is dissolved in oil (triglycerides), during oral administration, the suspension of particulates with broad size distribution would likely form [73] [36] [74] . Such suspension, with its natural tendency to coalescence, may phase separate leading to the delayed release from the stomach and possible interference with the foodstuff J o u r n a l P r e -p r o o f [79] . The other factor that needs to be considered is the enzymatic hydrolysis of the oily solution, which will alter the vitamin D 3 /oil ratio. Since vitamin D 3 solubility in oil is limited, the progression of hydrolysis may cause vitamin precipitation, making its absorption unlikely [75] . Based on the abovementioned facts, one can propose the fate of the oily formulation of vitamin D 3 in the gastrointestinal tract. Specifically, the oil with vitamin D 3 will form a heterogeneous emulsion with particulate sizes ranging from submicron up to hundreds of microns in diameter [80] . The emulsion being inherently unstable would tend to form a separate phase in the stomach, delaying its transfer to the intestine, hence slowing and/or limiting the absorption. A similar analysis may provide functional and topological requirements for the effective vitamin D 3 delivery vehicle. Specifically, the vehicle in the form of preformed suspension of stable particulates with well-defined sizes should be beneficial. Liposomes satisfy all these requirements. They can be prepared in such a way so the stable suspension of small vesicles can be formed [81] . The technologically available sizes of liposomes match the physiological requirements imposed by mucus and endocytic J o u r n a l P r e -p r o o f Journal Pre-proof processes. They also do not coalesce, so their size in the gastrointestinal tract will not increase. More importantly, the action of bile will rather reduce their sizes, therefore further accelerating their absorption. Consequently, it can be predicted that liposomes with vitamin D 3 will be internalized without delay after entering the intestine. In summary, the active hydrophobic compound will stay in the gastrointestinal tract in two forms: as a precipitate or solubilized in nonpolar solvent, i.e. fats [27] . It can be absorbed only when it is dissolved in the nonpolar phase, which prevents its precipitation. In such a case, the digestion process can be considered as the evolution of the two-phase system, oil/water emulsion or liposome suspension. The resulting particulates are altered by mechanical agitation, enzymatic transformation, and modification by detergents (bile acids). The detailed analysis presented in the paper leads to the conclusion that to achieve efficient absorption of hydrophobic compounds such as vitamin D 3 , the preformed, stable in time, particulate suspension such as liposomes with a well-defined size distribution should be used. The preliminary clinical experiment shows that while the liposomal vitamin D 3 formulation delivered orally elevates rapidly calcidiol concentration in the serum, no such effect was observed when the oily solution was used. In addition, the clinical experiment shows for the first time that vitamin D 3 intake may depend on the patient status with respect to the vitamin D 3 level. 

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