gentamicin encapsulation in liposomes: factors affecting the efficiency

Journal of Liposome Research 2(1), 11-22 (1992)

Gentamicin Encapsulation in Liposomes: Factors Affecting the Efficiency

Yolanda Cajal,' Maria Asuncion Alsina,* Maria Antonia Busquets,' Ana Cabanes,' Francisca Reig,' and Jose Maria Garcia-Anton' Salgado, 18-26, 08034 Barcelona, Spain; 2Physicochemical Department, Faculty of Pharmacy, University of Barcelona

'Laboratory of Peptides, C.I.D., C.S.I.C., Jordi Girona

Gentamicin was encapsulated in small unilamellar vesicles (SUV), multilamellar vesicles (ML V), and dehydration-rehydration vesicles (OR V). The encapsulation efliciency o j the three liposomal preparations was compared. The values corresponding to DRV are ten to fifteen times greater than those for ML V or SUV. For dehydration-rehydration vesicles, the influence of lipid constituents, mixtures of PC, PS, SA, Chol, in encapsulation efficiency was also determined. The stability of the DR V preparations yielding the maximum encapsulation value, as a function of osmotic gradients and incubated in the presence of blood, was determined.

Address reprint requests to: Dr. Francisca Reig, Laboratory of Peptides, Department of Bio- logical Organic Chemistry, Jordi Girona 18-26, 08034 Barcelona, Spain.

11

Copyright 0 1992 Marcel Dekker, Inc.

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INTRODUCTION

Since they were first reported in 1965 (l) , liposomes have been widely con- sidered as potential drug delivery systems (2). The accumulated evidence has confirmed their usefulness in avoiding some practical problems raised by the administration of different drugs. Thus, it has been reported that the toxicity of many therapeutic drugs (3-5) can be significantly reduced when administered in conveniently designed liposomes. In addition, adsorption (6,7), bio- distribution, and pharmacokinetics (8) of many drugs may be avoided or im- proved by administering them encapsulated in liposomes.

Nevertheless, in the case of drugs that have to be administered in relatively high doses there are still problems related to the encapsulation efficiency of the liposome preparations. Moreover, the entrapment efficiency of drugs in liposomes is highly dependent on the combination of a series of factors such as the molecular structure of the drug, the lipid composition of the liposomes, and the liposomes’ preparation method. For this reason, there is no general picture or rule concerning the entrapment efficiency of a drug in liposomes.

Aminoglycoside antibiotics have been studied in liposome preparations (9- 12), but to the best of our knowledge the low entrapment levels obtained make it difficult to assess the pharmacokinetics of these systems due to the high volume necessary to achieve therapeutic dosages.

Accordingly, in order to improve the entrapment yield of aminoglycoside antibiotics and, particularly, gentamicin in liposomes, we examined some of the factors affecting the entrapment efficiency. The influence of three different liposome preparation methods (MLV, SUV, and DRV) as well as five liposome compositions was studied. In addition, the stability of these preparations was tested in the presence of buffer solutions of different osmolality and in the presence of blood,

MATERIALS AND METHODS

Chemicals

Gentamicin sulphate (Danish Powder & Tabletting Factory Ltd. ApS, the labeled potency being 638.4 U/mg of dry powder) was kindly supplied by In favet. o-Phthalaldehyde (OPA) was purchased from Scharlau. Disodium hydrogen phosphate dodecahydrate, mcnosodium hydrogen phosphate di- ihydrate, sodium chloride, and trichloroacetic acid were from Merck (analytical grade). Lipids used were: egg phosphatides (Powder type 5) (PC), supplied by Asahi Chemical Industry; quantitative phosphate analysis was carried out by following the method of Marshall Stewart (13), and the phosphatides’ content was higher than 99%. Cholesterol (Chol) (Panreac), L-a-phosphatidyl-L- serine (PS), from bovine brain (Sigma) and stearylamine (SA) (Sigma); their

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GENTAMICIN ENCAPSULATION IN LIPOSOMES 13

purity was assessed by TLC on DC-Alufolien Kieselgel 60 (Merck), eluted with a solvent mixture of H,O/Cl,CH/MeOH (4:65:25) and one single spot was visualized in each case after spraying with perchloric acid 40% and charring.

PBS solution contained 29 g of disodium hydrogen phosphate dodecahydrate, 2.6 g of monosodium phosphate dihydrate, and 2.8 g of sodium chloride in 1 L of distilled water (pH = 7.42). Its conductivity was 15.095 ms/cm.

Multilamellar vesicles (ML V) were prepared as follows: samples of 25 mg of PC and 12.5 mg of cholesterol were evaporated from a chloroform-methanol (2: 1 v/v) solution. The samples were submitted for 2 hours to the vacuum of an oil pump to eliminate better the last traces of solvent. Two ml of gentamicin solution in PBS (1 mg/ml) was added to the dry lipid, and the suspension gently stirred during 1 hour at room temperature (21°C).

Small unilamellar vesicles (SUV) were obtained by sonication of a cooled multilamellar vesicles preparation with a probe sonicator under an inert at- mosphere of nitrogen.

Dehydration-rehydration vesicles (OR V) were prepared according to the de- scription given by Kirby and Gregoriadis (14). Lipid quantities were the same as for MLV, but the dry lipids were rehydrated with 2 ml of water, and soni- cated to obtain SUV. Then, the preparation was contrifuged at 1700 x g to remove titanium particles released from the sonication probe. To 1 ml of this preparation, 500 pl of gentamicin solution (2 mg/ml) was added and the mixture freeze-dried. The remaining residue was rehydrated with distilled water (0.1 ml) and PBS (0.9 ml), and finally 7 ml of PBS were added.

After preparation, MLV and DRV liposomes were washed three times by successive centrifugation at 25,000 x g and resuspension of the pellets in PBS. SUV were separated from the non-encapsulated material by dialysis (1 : 10oO v/v) for 8 hours.

Gen tam k i n Quantification

The gentamicin content of washing supernatants and liposomes was determined by HPLC following a standard OPA precolumn derivatization pro- tocol (15-17). To 300 pl of liposomes (PC/gentamicin: 12.5:l w / ~ ) , 300 pl of isopropanol were added and then derivatized by adding 300 pl of OPA reagent. This reagent had been prepared by dissolving 50 mg of OPA in methanol (1 ml), and adding 40 pl of 2-mercaptoethanol and 9 ml of potassium borate buffer (pH= 10.5). After 15 minutes of reaction, samples were taken and analyzed on a Merck-Hitachi HPLC system fitted with a Lichrospher 100- RP-18 (5 pm) column, eluted isocratically with a mixture of methanol/acetic

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14 CAJAL ET AL.

acid 10% solution (77:23, v/v) containing 4.5 g/L heptanesulfonic acid sodium salt, at a flow rate of 1 ml/min.

The eluted substances were monitored with a fluorescence detector (Perkin- Elmer, model LS-1) working at 340 nm of excitation wavelength and using a kv 418 nm filter for detection of the emission. The amounts of gentamicin were calculated from peak areas by the external standard method on a D-2000 Hitachi-Merck chromatointegrator. This method allows the detection of 0.3 pg/ml of gentamicin.

Stability Studies

The stability of DRV liposomes containing encapsulated gentamicin was tested in the presence of PBS solutions of the following conductivities: 15.095 ms/cm (PBS standard), 8.618 ms/cm (PBS diluted 1:2), and 4.646 ms/cm (PBS diluted 1:4) and in the presence of blood.

Stability in Iso-Osmotic and Hypotonic Conditions

Liposomes to be tested were previously centrifuged at 14,000 rpm during 25 minutes; the pellet was washed twice to remove non-entrapped material and finally diluted with PBS (standard) to 10 ml. This preparation contained 1.25 mg PL/ml and 0.064 mg gentamicin/ml.

From this solution aliquots of 1 ml were added to 9 ml of PBS of the desired concentration and stored at 4°C. Samples of 0.5 ml were taken every 7 days, centrifuged, and the supernatants derivatized to quantify the gentamicin content by HPLC.

Stability In Vitro in Presence of Blood

Gentamicin-containing liposomes (400 p1) (1.25 mg PC/ml and 64 pg gentamicin/ml) were added to 1 ml blood (triplicate) in a polypropylene micro- test tube, and the mixture incubated at 37°C. At time intervals samples were taken by triplicate and processed as follows. Blood cells were removed by centrifugation at 3000 rpm (15 min, 4°C). A 500 p1 aliquot of the supernatant was centrifuged at 15,000 rpm (10 min) in order to pellet the liposomes. The supernatant was deproteinized by addition of trichloroacetic acid (40%) and subsequent centrifugation at 15,000 rpm (10 min). The protein-free supernatant was neutralized with 25 pl sodium hydroxide (20% w/v). Derivatization and quantification by HPLC was carried out as for aqueous gentamicin solutions.

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RESULTS AND DISCUSSION

Accuracy of the Analytical Method

Gentamicin analysis in water solutions and in biological fluids has been ex- tensively reviewed in the literature (18-21). Nevertheless, as far as we know, there are no papers about the recovery and quantification of this drug when encapsulated in liposomes. For this reason we checked previously that liposomes were destroyed before derivatization and also that the presence of phospholipids and cholesterol does not mask the gentamicin content of the preparation. As far as the total gentamicin recovery from liposomes is concerned, it can be concluded that it is not necessary to extract lipids with chloroform treatment, if during the derivatization process samples were heated for 5 minutes after isopropanol addition and before OPA reaction.

Concerning the effects of the presence of phospholipids, two standard preparations containing the same amount of gentamicin but with one of them added to some amount of void liposomes had been quantified. The HPLC results fall in the same range. These facts allow us to be sure of the results obtained when quantifying entrapment and stability characteristics of the different liposome preparations.

Moreover, to compare the usefulness of dialysis or centrifugation to eliminate non-entrapped material, a preliminary study of the gentamicin dialysis kine- tics was carried out, working with the same drug concentration that would exist in liposome preparations. Two sets of experiments were done dialyzing free gentamicin 250 pg/2 ml against 2000 ml PBS for 8 hours or 250 pg/2 ml against 50 ml PBS with changes every 2 hours (4 changes).

The gentamicin content of the external phase was very similar in both cases, and after 8 hours never reached 70% of the initial amount. This fact prompted us to use mainly centrifugation for removing non-entrapped material.

Encapsulation of Gentamicin as a Function of Liposome Size

MLV, SUV, and DRV were prepared as described in the experimental part. Lipo- somes were separated from non-entrapped material by dialysis in the case of SUV and by centrifugation or dialysis in MLV and DRV. In some cases “basket centrifugation” (22) was assayed for SUV. The gentamicin content in the supernatants was also determined in order to check if some losses of drug were pro- duced. Each liposomal preparation was done in triplicate and submitted to at least two independent derivatizing processes and subsequent HPLC analysis.

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16 CAJAL ET AL.

The entrapment and encapsulation efficiencies given as mg gentamicin/mmol PL and percentage of the initial amount of drug incorporated into the liposomes are given in Table 1. The composition was the same in all cases: PC/ Chol(1: 1). The figures in Table 1 show clearly that DRV’s liposome preparation method gives higher encapsulation levels than MLV or SUV.

Another interesting point relates to the method followed to separate free from entrapped drug. Several authors prefer to avoid centrifugation processes to preserve better the integrity of their preparations. In these cases dialysis can be an alternative procedure to remove non-entrapped material. To compare which one of these methods renders better results in our system, after rehy- drating DRV, preparations were divided into two parts and submitted either to dialysis or centrifugation. When using centrifugation, liposomes were washed three times with PBS and the gentamicin content of liposomes and the three consecutive supernatants was analyzed. The results show that the washing process was nearly completed after two washings, the gentamicin content in the third supernatant being nondetectable. Furthermore, the total content of gentamicin in the pellet and in the supernatants was close to the initial amount of gentamicin used to prepare the liposomes. This result indicates that this method permits the liposome preparation to be completely free of non-encapsulated material. The second lot of liposornes was submitted to dialysis and the calculated encapsulation efficiency was lower.

This difference between the two procedures cannot be attributed to the leakage of encapsulated gentamicin during dialysis because stability studies show that this process is very slow. In our opinion there may be some adsorption of liposomes on the dialysis bag walls that could explain these small differences.

Table I. Incorporation of Gentamicin into MLVs, SUVs, and DRVs (Composition PC/Chol 1:l).

Encapsulation Entrapment Efficiency Efficiency

Liposomes (YO) (mg/mmol PL)

MLV 7.20 f 1.00 (a) 4.55 f. 0.63 (a) suv 4.33 f 0.47 (b) 2.74 f 0.29 (b)

0.55 f 0.06 (c)

DRV 64.38 k 2.54 (a) 40.74 f 1.61 (a) 57.41 f 2.06 (b) 36.33 f 1.30 (b)

0.88 k 0.10 (c)

Method to separate free from entrapped drug: (a) centrifugation; (b) dialysis; (c) basket centrifugation.

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SUV liposomes can be separated from non-encapsulated gentamicin on mini- columns of Sephadex G-25 made from the barrels of 5 ml plastic syringes (“basket centrifugation”). The column was inserted into a test tube so that it was supported at the top of the tube by the finger grips of the syringe and spun at 3000 rpm (1500 g) for 10 minutes in a Beckman model TJ-6 centrifuge to remove excess fluid from the Sephadex beads. The syringe was transferred to another test tube and up to 1 ml of the liposomal preparation containing entrapped and free solute was applied to the Sephadex bed. The column was spun at 1000 rpm (200 g) for 1 minute and then at 3000 rpm (1500 g) for 10 minutes, expelling the liposomal material from the column and into the test tube.

This method is faster than dialysis, but the recovery of SUV liposomes, with only one centrifugation of the column, was incomplete. Thus, for quantitative determinations the better methodology was dialysis.

Stability of Liposomes

As far as their future therapeutic application is concerned, the stability of liposome preparations is a very important factor. In the present study we have determined the gentamicin leakage from DRV-liposomes (composed of PC/ Chol 1 : l), maintained in iso-osmotic conditions or suspended in hypotonic PBS solutions (PBS concentration equal to 1/2 or 1/4 of the standard solution). The gentamicin content of the supernatants (Figure 1) is given as a function of time and represents the average of three independent preparations. Drug leakage was calculated as the percentage of released drug towards the initially encapsulated.

At first, one can appreciate that these liposomes are very sensitive towards osmotic gradients. The ones suspended in the presence of hypo-osmotic buf- fers are more permeable to gentamicin than the liposomes stored in PBS of standard concentration. These results are in agreement with the CF perme- ability measured by Kirby and Gregoriadis (14).

Moreover, our results show that during the first week there is an important leakage of gentamicin, but afterwards the process advances very slowly. Prob- ably the diffusion of gentamicin molecules across the bilayer tends to reach an equilibrium value, when concentrations in and out become more similar.

As far as the stability in blood is concerned, due to the complexity of gentamicin isolation and quantification in these experiments, each point represents the average of three determinations.

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18 CAJAL ET AL.

t 00

90 -

00 -

70 - 9) rn rd .Y

1 2 3 4

t (weeks)

Figure 1. Stability of DRV liposomes in PBS of different osmolality. C, PBS; 0 , PBS (1:2); A, PBS (1:4). Each point is the mean k S.D. of three independent preparations.

Table 2. Stability of DRVs Incubated at 37°C in the Presence of Blood

Time (min) Gentamicin Leakage (Yo)

0 1.38 f 0.24 15 3.80 f 0.32 30 4.35 * 0.21 60 4.41 k 0.10 90 4.98 f 0.34 120 6.21 f 0.86

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The results obtained (Table 2) show that DRV liposomes containing gentamicin are highly stable in blood at 37°C for at least 2 hours. Only 6% of leakage was observed after 120 min of incubation. These values are in agreement with the ones published by Senior (23) for CF. In this case, the most important factor is probably the high content of Chol, which reduces the phospholipid removal by the HDL of blood.

Encapsulation of Gentamicin as a Function of the Liposome Chemical Composition

The influence of cholesterol and anionic or cationic lipids in the encapsulation level of gentamicin in DRV was studied by preparing liposomes of varying composition. The entrapment and encapsulation efficiency were checked by centrifugation and resuspension of the pellets and also by dialysis. The values are given in Table 3,

In all cases it is clear that the dialysis process provides an incomplete recovery of the product.

Moreover, in this system the presence of ionic charges seems not to be crucial to improve the encapsulation efficiency of the preparations. The lower values for liposomes containing 10% PS were the opposite expected, having in mind

Table 3. Composition

Incorporation of Gentamicin into DRVs of Varying Lipid

ComDosition

Encapsulation Efficiency

(070)

PC 0.53 f 0.06 (a) 1.06 f 0.14 (b)

PC/Chol (2: 1) 17.58 f 0.89 (a) 16.79 f 0.53 (b)

PC/Chol (1 : 1) 64.38 f 2.54 (a) 57.41 f 2.06 (b)

PC/Chol/PS (1 : 1 :0.2) 47.04 f 0.42 (a) 42.68 f 1.36 (b)

PC/Chol/SA (1 : 1 :0.2) 62.26 k 8.16 (a) 61.38 +- 0.58 (b)

Entrapment Efficiency

(mg/mmol PL)

0.22 k 0.02 (a) 0.45 f 0.06 (b)

11.12 f 0.56 (a) 10.63 f 0.33 (b) 40.74 f 1.61 (a) 36.33 f 1.30 (b) 29.77 f 0.26 (a) 27.01 f 0.08 (b) 39.40 f 5.16 (a) 38.84 k 0.37 (b)

Method to separate free from entrapped drug: (a) centrifugation; (b) dialysis.

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20 CAJAL ET AL.

the positive ionic charge of gentamicin molecules. Neither could we find any explanation for the poor results obtained with pure PC. The only hypothesis we can propose is that due to the saturated character of PC used in these assays and the higher transition temperatures associated with these kind of phospholipids (in spite of the fact that they contain different chain lengths), it would be necessary to rehydrate at higher temperatures. If this is true, the presence of cholesterol, which regulates the fluidity of the bilayer, would render better encapsulation efficiencies working at room temperature as it is in this case.

The results obtained in the present paper confirm the fact that it is not pos- sible at the moment to generalize about encapsulation and separation methods. In each case it is necessary to review a previous accurate study to find the best working conditions.

Moreover, as Kirby and Gregoriadis suggested, in our hands DRV proved to be one of the best methods to achieve high encapsulation levels, which could improve the usefulness of liposome preparations as delivery systems.

ACKNOWLEDGMENTS

This work was partially supported by Infavet and by a grant (PAM-01 10-C 02-02) from DGICYT (Spain). One of the authors is a recipient of an FPI grant from the Ministerio de Educacion y Ciencia (Spain).

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gentamicin encapsulation in liposomes: factors affecting the efficiency

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