Liposome encapsulated vitamin A compounds exhibit greater stability and diminished toxicity

Anil K. SinghU , Joydip Das

Abstract Absorption and uorescence studies of retinol vitamin A alcohol. and retinol palmitate vitamin A palmitate. intercalated in phosphatidylcholine PC. liposomes show that these compounds are bound to the lipid bilayer. It is further found that retinol binds liposomes with greater afnity as compared to retinol palmitate. In addition, the delivery of liposome-incorporated retinoids to the blood has also been studied and it is found that these systems reduce blood viscosity and cause less lysis of red blood cells than retinoid compounds not complexed in liposomes.

Keywords: Liposome; Retinol; Retinol palmitate; Absorption; Fluorescence; Binding; Viscosity; Lysis; Delivery

1. Introduction Inspite of much biological and pharmacological effects, the therapeutic use of retinoids Fig. 1. is still limited by reasons of many adverse effects that characterize the activity proles of retinoids, especially when topically employed at higher doses or administered systematically w1 9x. The failure to adopt synthetic retinoids and their parent compounds, such as retinol, retinoic acid and the corresponding esters for clinical use is mainly due

to strong toxicity hypervitaminosis., poor chemical stability, long-lasting tetratogenicity, etc. In addition, the hydrophobic nature of retinoids makes their administration difcult by either intravenous or oral routes, requiring the utilization of oily formulations or surfactant containing aqueous solutions. In the past, liposomes and immunoliposomes referred to as biological couriers or biological missiles. have been suggested as possible pharmaceutical delivery systems w10x. Production, characterization and antiproliferative activity on neoplastic cells by liposome-associated retinoids have been reported w11x. Penetration of spinlabelled retinoic acid from liposomal preparation

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Fig. 1. Chemical structures of vitamin A compounds. 1. Retinol vitamin A alcohol.; 2. retinol palmitate vitamin A palmitate; 3. retinoic acid vitamin A acid.; 4. retinal vitamin A aldehyde..

into the skin of SKHI hairless mice has been studied by EPR tomography techniques w12x. Earlier we have demonstrated that liposomes can stabilize vitamin A compounds w13,14x. With the aim of nding ways to administer retinoids, possibly overcoming or alleviating the solubility, specicity and toxicity problems associated with retinoids use, we have studied the properties of retinol and retinol palmitate intercalated in phosphatidylcholine PC. liposomes. Liposomal vitamin A compounds have been characterized by absorption and uorescence spectroscopy, and biocompatibility and lysis effects of liposomal vitamin A compounds have been evaluated. It is found that retinol binds more efciently to liposomes as compared to retinol palmitate. Furthermore, liposome-associated retinoids cause less lysis of RBC membranes, and these are also found to reduce the viscosity and rigidity of RBC solutions. 2. Experimental details 2.1. Materials and general procedures Retinol, retinol palmitate and retinal were purchased from Fluka. The L--phosphatidylcholine was from Sigma. All spectroscopic studies were done in the UV-vis grade solvents procured from Mrs Spectrochem, Mumbai. Fresh human blood samples of blood group A q , B q and O q were obtained from the Department of Biomedical Engineering, I.I.T, Bombay. All other chemicals were from the Aldrich Chemical Company, USA and used as received. Double distilled and de-ionized

water was used for sample preparations. All the retinoids were handled in dim light red. conditions, and if required were stored in sealed vessel as n-hexane solution in a nitrogen atmosphere. Their stereochemical purity was checked by HPLC analysis w15x and only all-trans isomers were used in the present studies. UV-vis measurements were carried out on a Hitachi-U2000 spectrophotometer. Steady state uorescence studies were performed on a Spex Spectrouorolog instrument equipped with a 16810.22-m spectrometer as the single-grating monochromator, a 16800.22-m double spectrometer as the double-grating emission monochromator and a 450-W xenon lamp as the light source. Samples were adapted to normal room temperature 25C. prior to measurements. Fluorescence lifetime measurements were done on an Applied Photophysic SP-70 nanosecond spectrouorimeter equipped with a 250-W xenon lamp and 200 KHz nanosecond ash lamp as a light source. The lifetime was recorded using the single photon counting technique and data were processed by deconvolution methods. Excitation was done at 325 nm and emission was monitored at 495 nm. A PHM-84 Research pH meter from Radiometer, Copenhagen was used for measuring the pH values. Sonications were carried out with Branson B-12 sonier output power 400 W, frequency 20 KHz. equipped with microprobes. Centrifugations were done in Beckman L8-55M Ultracentrifuge using a 45Ti rotor. Viscosity measurements on blood samples were made in Contraves 30 Low Shear Viscometer and by following the literature procedure w16,17x. All operations related to retinoids were carried out in a dim light red. condition. 2.2. Preparation of retinoids constituted liposomes Incorporation of retinoids in liposomes was done as described earlier w14x. In a typical procedure, a chloroform solution of retinoid 20 l, 1.30 = 10y3 M. was mixed with a n-hexane solution of L--phosphatidylcholine 0 200 l, 2.5 = 10y2 M.. wA chloroform solution of cholesterol 2.00 7.00 = 10y5 M. was added in case of lipo-

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somes containing cholesterol.x The mixture contained in a round-bottomed ask was diluted with 2 ml of chloroform. Most of the organic solvent was removed from the mixture on a rotary evaporator under diminished pressure at 4C. The mixture was further kept under vacuum 10y4 torr. for 3 h to ensure complete removal of organic solvent when a thin shiny lm of the residual material is formed in the ask. The desired buffer 2 ml. of particular pH was then added to the ask and the mixture was kept at 4C for 3 h allowing the phospholipid to swell. The solution was sonicated for 7 min and then centrifuged at 5400 g for 20 min at 4C. Liposome-associated retinoids were further subjected to gel permeation chromatography over Sephadex G-25 with corresponding buffers as eluants. The liposomal preparations of retinoids thus obtained were kept under an argon atmosphere in the dark for further studies. 2.3. Fluorescence studies From ethanolic stock solutions 5.27 = 10y3 M. of retinol and retinol palmitate, a 4- l solution was taken and added separately to 2 ml PC solution with varying lipid concentration 0 2.0 = 10y4 M. in different glass vials. The liposomal solutions were incubated at room temperature for 20 min and the uorescence spectra were recorded. From the values of the change in intensity of emission maximum, the binding constants were calculated according to the literature procedure w18x. For characterizing the microenvironment of retinoids in liposome, quenching experiments were performed by addition of 4 l of a concentrated ethanolic solution of retinal 2.50 = 10y3 M. to the liposome-associated retinol palmitate. The excitations were done at 325 nm. 2.4. Studies with red blood cells Fresh human blood samples of blood group A q , B q and O q , obtained from the Department of Biomedical Engineering, I.I.T, Bombay, were centrifuged at 4000 rev.rmin for 5 min. The upper plasma and white buffy coats were removed by a syringe and RBCs remained in the lower

portion were washed thrice with water and with saline 0.9% NaCl in water.. A sample of 1% blood suspension was made by diluting it with phosphate buffered saline w19x pH, 7.2.. The retinoids 3.3 = 10y5 M. were added and incubated at 37C with gentle shaking. The extents of lysis of the RBC were determined by the increase in absorption of haemoglobin at 540 nm at different time intervals. Biocompatibility investigations were done by addition of retinol or retinol palmitate to RBC solutions or to whole blood and measuring the viscosity at different time intervals. Control experiments were performed following similar experimental protocols. 3. Results and discussion 3.1. Binding of retinol and retinol palmitate with PC liposomes: absorption and uorescence studies The UV-vis absorption characteristics of retinol and retinal palmitate are similar in the ethanol and liposomal matrix Table 1.. The absorption U band at approx. 326 nm is due to , transition of the retinoids polyene chain. The binding of retinol and retinol palmitate to PC liposomes has been determined by spectrouorimetric analysis. Excitation of liposome-bound retinoids retinol and retinol palmitate at 325 nm resulted in uorescence bands with max at 490 nm Fig. 2.. The uorescence intensities of retinol and retinol palmitate increase with the increase in phospholipid concentration with a xed concentration of retinoids. Typical uorescence intensity variation with increasing lipid concentrations for retinol are shown in Fig. 2. Similar variations are observed for retinol palmitate also. The binding constants of retinoids with liposomes. have been deTable 1 UV-vis absorption maxima of retinol and retinol palmitate Compound

ma x , nm .
Ethanol Liposomea 328 27,657. 330 32,339.

Retinol Retinol palmitate

325 52,995. 327 51,177.

Phosphatidylcholine, 5 = 10y4 M.

158 Table 2 Binding constant K , My1 . of retinol and retinol palmitate with liposomes Compound Retinol Retinol palmitate
a

K My1 .a 3.61 = 104 1.03 = 104

An average of three sets of experiments; error ; "3%.

1 termined by a plot of r a y 1.y1 vs. Cy 1 ; where s uorescence quantum yield of retinoid in liposome, a s uorescence quantum yield of retinoid in aqueous solution, K s binding constant, and C1 s phospholipid concentration wMx. 1 A typical plot of r a y 1.y1 vs. Cy for retinol 1 is shown in Fig. 3. A similar straight-line plot was obtained for retinol palmitate also. The slope of these straight-line plots gave the K-values Table 2.. The high binding constant values of the order of four indicate efcient binding between retinoids and liposomes. The K-value of retinol is higher as compared to the K-value of retinol palmitate. This is due to the presence of relatively more polar hydroxyl group in retinol which can interact more with the polar head groups of phospholipid than does the relatively less polar ester group of retinol palmitate. Fluorescence quenching experiments and uo-

Fig. 3. A plot of ra y 1.y1 vs. wphosphatidylcholine, PC, Mxy1 , for retinol in PC liposomes.

rescence lifetime measurements of retinol palmitate have enabled us to further characterize the binding site of retinoids in a liposomal matrix Table 3. The all-trans-retinal quenches the uorescence of liposomal retinol palmitate. The corresponding Stern Volmer plot w20x for this quenching experiment is shown in Fig. 4. Retinol palmitate in liposomes shows a uorescence lifetime of 2.17 ns and the corresponding uorescence decay prole is presented in Fig. 5. The Stern Volmer constant K sv and the microviscosity . of the localization domain for liposomal retinol palmitate is found to be 6.73 = 10 4 My1 and 3.22 = 10y1 3 N s my2 , respectively. These values indicate that retinol palmitate resides in a rather less viscous and less hydrophobic liposomal domain. This becomes obvious when we compare the K sv and values of
Table 3 Fluorescence characterization data for retinol palmitate in the liposome Parameter Value 6.73 = 104 My 1 2.17 ns 3.10 = 1013 My 1 sy 1 3.22 = 10y 13 N s my 2

Fig. 2. Fluorescence spectra of retinol in phosphatidylcholine PC. liposomes. PC concentrations are wax, 0.0 M; wbx, 5 = 10y6 M; wcx, 1 = 10y5 M; wdx, 5 = 10y5 M; wex, 1.0 = 10y4 M; wfx, 2 = 10y4 M. Excitation at 325 nm.

Stern Volmer constant, Ksv Fluorescence lifetime, 0 Quenching constant, kq Microviscosity,

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Fig. 4. Stern Volmer plot for quenching of retinol palmitate uorescence by retinal. Excitation at 325 nm.

the molecule is hydrophillic or hydrophobic, it could be incorporated in the entrapped aqueous interior or in the lipid bilayer. The hydrophobicity of a molecule could be judged by its polarity. Between retinol and retinol palmitate, the former is more polar than the later. It is observed that in reverse phase HPLC C 18 . retinol elutes faster than retinol palmitate w22x. The length of retinoids can be calculated from the energy minimized structures using SYBYL Tripos, St Louis, MO, USA. and it is found that retinol and retinol palmitate, respectively, have lengths of 15.5 and and steric energy of 41.64 and 58.18 kcal 35.8 A, moly1 . Thus, retinoids can easily be accommodated in the lipid bilayer as the thickness of the Thus, retinoids can be bilayer is approx. 40 A. stabilized in the lipid bilayers of liposomes. 3.2. Effect of liposomal and non-liposomal retinol and retinol palmitate on red blood cells (RBC) Effect of liposomal and non-liposomal retinol and retinol palmitate on RBC membranes has been investigated by monitoring the erythrocytes absorption band at 540 nm. As seen in Fig. 6, retinol has a drastic lytic effect on human RBCs. In contrast, retinol palmitate causes little or no

retinal-caused uorescence quenching of hydrophobic pyrene which is expected to localized in a hydrophobic domain of the liposome. Liposomes are spherical, self-closed structures composed of curved lipid bilayers which entrap part of the aqueous media in which they freely oat into their interior. The thickness of the w21x. Depending on whether bilayer is approx. 40 A

Fig. 5. Fluorescence decay prole of retinol palmitate in PC liposomes.

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Fig. 7. A plot of change in absorbance at 540 nm with time for incubation of retinol in a. phosphate buffered-saline; b. PC liposomes; c. PC liposomes with erythrocyte at 37C.

Fig. 6. A plot of change in absorbance at 540 nm with time for incubation of a. retinol; b. retinol palmitate; c. control with erythrocyte in phosphate buffered saline at 37C. Inset: absorption spectrum of erythrocyte after lysis by retinoids.

lysis at the concentrations used in the present experiments. Liposomal retinol palmitate lysis data are not shown, since the reduction in lysis for liposomal and non-liposomal retinol palmitate is very less as compared to the liposomal and non-liposomal retinol. Control experiments were done without adding any retinoid and it was found that lysis of the cells does not occur. The lytic effects of retinol may be due to the polarity of its functional group. When retinol was incorporated in PC or in PCrcholesterol liposomes w14x and incubated with human RBCs, it caused lysis to a lesser degree Fig. 7.. The lytic effect of the two types of liposomes are, however, comparable. This study shows that retinol, unlike retinol palmitate, can interact strongly with natural membrane. The initial action of retinol on RBC can be a penetration and expansion of the cell membrane as evidenced by observations from a sequence of changes in the ne structure of the cells during lysis by the vitamin w23x. However, when retinol is incorporated in liposomes, only a

small amount of lysis occurs; presumably the liposomes can sequester the drug and prevent its interaction with erythrocytes. At present the mechanism underlying the reduced toxicity of liposomal retinoids is not very clear. It is well known, however, that incorporation of drugs into liposomes can affect its behaviour in vivo in a variety of complex ways including alteration of pharmacokinetics, changes in tissue distribution and modication of immune functions. 3.3. Biocompatibility of liposomal retinol palmitate effect on blood iscosity Biocompatibility of liposomal retinol palmitate has been investigated by measuring the viscosity changes of RBC and the whole blood. The effects of retinol palmitate and liposome-associated retinol palmitate on erythrocytes viscosity as a function of shear rate is shown in Fig. 8. In Fig. 9 the effects of liposomal and non-liposomal retinol palmitate on the viscosity of the whole blood at a shear rate of 95.5 sy1 are depicted. It is found that the viscosity is reduced in the presence of retinol palmitate and is further reduced in the presence of liposome-associated retinol palmitate. The decrease in viscosity is more at a low shear

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Fig. 8. Change of viscosity with shear rate. a. Erythrocyte; b. erythrocyte and retinol palmitate; c. erythrocyte and PC-liposomal. wRetinol palmitaex, 3.3 = 10y5 M; wphospholipidx 4.15 = 10y4 M.

Fig. 9. Change of viscosity with incubation time for whole blood at a shear rate of 94.5 sy1 for a. whole blood; b. retinol palmitate in whole blood; c. liposomal PC.-retinol palmitate in whole blood. Retinol palmitate 3.3 = 10y5 M; phospholipid 8.30 = 10y4 M.

rate. It is possible that in the presence of an external lipid, the lipids of RBC get exchanged with the former, and as a result, the rigidity of the red cells decreases. Consequently, decrease in the viscosity is observed. The red cell membrane bilayer comprise mainly of phospholipids and cholesterol with a small amount of glycolipids and membrane proteins. The length and degree of saturation of fatty acid residues of the phospholipid strongly inuences the uidity of the membrane. With increasing chain length and degree of circulation, the fatty acids become less uid and the membrane becomes more viscous w24x. 4. Conclusions In conclusion it can be said that liposomes can be used as a retinoid delivery system which can also reduce the toxicity of the retinoids. This approach of intercalating vitamin A compounds in liposomes provides a means of increasing the much desired stability and structural and functional specicity of retinoids and may lead to the further development of other novel systems for use in vitamin A therapeutics. We have not shown how the retinoids could be released from the liposomes. In this context, it may be mentioned that recently the rate of transfer of radioactive

retinoids from vesicles to erythrocyte has been studied and it is found that inter-membranous transfer of retinol is much faster than the retinyl palmitate w25x. This is because the long fatty acyl chain in the latter makes it more hydrophobic and since the long chain fatty acyl retinyl esters are not transferable at substantial rates, putative binding proteins for these hydrophobic derivatives would be expected to play a catalytic role in inter-membranous transfers. The targeted release could be achieved by making immuno-liposomes, i.e attaching antibodies with the liposomal retinoids, and further work in this area is highly warranted. Acknowledgements We thank the School of Biomedical Engineering and RSIC, I.I.T, Bombay for technical assistance and the Department of Science and Technology, and the Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India for grants to purchase some of the equipments used in these studies. JD gratefully acknowledges the receipt of research fellowship from the Council of Scientic and Industrial Research, New Delhi, Government of India. We are gratefully thankful to the reviewers of this paper for their valuable suggestions.

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