Liposomal Entrapment of Suramin

HUNG-CHIH CHANG AND DOUGLAS R. FLANAGAN~ Received January 5, 1994, from the Division of Pharmaceutics, College of Pharmacy, University of Zowa, Iowa City, ZA 52242. Accepted for publication March 17, 1994'.

Abstract 0 The liposomal entrapment of suramin and similar compounds in phospholipid vesicles was examined. For dipalmi- toylphiosphatidylchohe (DPPC) liposomes, entrapment percentages ranged from 25 to 65% with 3-25 mM phospholipid for aqueous solutions containing 0.07 mM of suramin. Incorporation of 30-50 mol % cholesterol (CHL) into DPPC liposomes reduced the percentage suraniin entrapment. Addition of positively-charged stearylamine (5 mol %,)to DPPCKHL liposomes increased the entrapment from 2.3 % to 30.3%. Entrapment was not affected by the incorporation of negatiively-charged phosphatidylglycerol into DPPCXHL liposomes. When the amount of suramin was increased from 0.07 to 0.7 mM, the entrapment percentage decreased from 37 % to 11 % when DPPC was held constant at 6 mM. The entrapment of 0.07 mM Evans blue, a mollecule similar in structure to suramin, was 51.6% in DPPC liposomes for 6 mM phospholipid. The entrapment percentage, however, decreased by about 50% when incorporated into 7:3 (DPPC/ CHL) Iliposomes. The liposomal entrapment of disodium 1 ,5-naph- thalenedisulfonic acid (5.5 %) and sodium 3-amino-2,7-naphthalene- disulfanic acid (1.2%) was very low compared to that of suramin or Evans blue. Differential scanning calorimetry studies of suramin and an aqueous dispersion of DPPC showed an apparent interaction between them. These observations suggest that a significant portion of the entrapped suramin results from binding of suramin to the surface of or intercalation into the liposomal bilayer. Surface binding or intercalation into the phospholipid bilayer may be attributed to both ionic and hydrophobic interactions. The Ionic interaction would arise from the suramin sulfonate groups associating with the cationic choline portion of the phospholipid. The hydrophobic interaction would arise from the central portion of the suramin molecule associating with the phospholipid fatty acid chains.

Introduction Liposomes are microscopic structures consisting of one or more

concentric lipid bilayers surrounding water entrapped from the environment. Liposomes are now considered as a drug-delivery system that can improve the therapeutic safety and activity of a coneJderable number of c0mpounds.1~~

Suramin is the hexasodium salt of a symmetrical bis- (naphthalenetrisulfonic acid). It has been used for treatment

NH NH

co co I NaO:,S I

of trypanosomiasis for over 50 years and has recently been studied for the treatent of cancer.4 In vitro, suramin has shown the ability to block the binding of a wide range of tumor growth factors5 such as platelet-derived growth factor, basic fibroblast growth factors, tumor growth factor-& and epidermal growth

0 Abiitract published in Advance ACS Abstracts, May 1,1994.

factor to their cell surface receptors, thereby antagonizing the ability of these factors to stimulate growth of tumor cells. Some clinical trials suggest antitumor activity of suramin at plasma concentrations of -200 pg/mL at doses of 0.85-1.2 glmziwk.4 Suramin is also the first reverse transcriptase (RT) inhibitor shown to suppress HIV replication in cell culture" and in AIDS patients.8 Several suramin-like compounds have been prepared which have an inhibitory effect on HIV RT.S-l2 However, the usefulness of suramin is limited by its rather high toxicity.8 Suramin is also unusual because its terminal plasma half-life is quite long (45-50 days) and its protein binding is very high (99.7% ).

The therapeutically effective suramin concentration (-200 pg/mL) may not be applicable to liposome-entrapped suramin since its distribution and protein binding will be altered. We hope that incorporation of suramin or similar compounds into liposomes will increase their delivery to HIV-infected lympho- cytes in AIDS or to solid tumors and decrease the required dose. Also, the rather high toxicity of suramin may be reduced by incorporation into liposomes, increasing its margin of safety. Since nothing is known about the liposomal entrapment ef- ficiency of suramin, we have investigated its incorporation and that of similar compounds into phospholipid vesicles.

Materials and Methods Dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol

(PG) were purchased from Avanti Polar Lipids (Birmingham, AL). Cholesterol (CHL), stearylamine (ST). and Evans blue (>95% purity) were obtained from Sigma Chemical (St. Louis, MO). Disodium 1,5- naphthalenedisulfonic acid and sodium 3-amino-2,7-naphthalene- disulfonic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Suramin was from FBA Pharmaceuticals. These lipids and chemicals were used without further purification.

Preparation of Liposomes-Initially, liposomes were prepared by the conventional thin-film hydration meth0d.13-~6 Various lipid amounts were dissolved in 10 mL of methylene chloride. This solution was rotary evaporated to form a thin lipid film on the wall of a 100-ml round- bottom flask. Residual solvent was removed by storing the flask under vacuum for 1 h. Five milliliters of suramin solution in normal saline and a few glass beads were added to the flask. The flask contents were subjected to further rotary evaporation at 50 OC until all the lipid was removed from the flask. The liposomal dispersion was a homogeneous milky white suspension which was then centrifuged (IEC CENTRA-7R centrifuge, 25 OC) at 4500 rpm for 30 min to separate free suramin. The supernatant solution was removed and 3 mL of normal saline added. The liposomes were redispersed and centrifuged with redispersion two more times. The supernatant solution aliquots were pooled for UV assay at 312 nm. After the final centrifugation, liposomes with entrapped drug were dissolved in a mixture of 2-propanol and water (3:l) for content analysis by UV assay. The entrapment percentage was calculated as

entrapped suramin total suramin entrapment percentage =

Liposomes prepared in this manner and examined visually by a laser scanning confocal microscope (Bio-Rad, MRC-600) were multilamellar (MLV) vesicles. Liposomal size distribution was determined by dynamic laser light scattering analysis using a Nicomp 270 submicron particle sizer.1618 The DPPC liposomes gave a bimodal volume-average size distribution: one centered at 0.97 p m (0.4%) and the other centered at 8.18 pm (99.6%). The larger distribution is a t the upper limit for the

0 1994, American Chemical Society and Americisn Pharmaceutical Association

0022-3549/94/ 1200- 1043$04.50/0 Journal of phermaceurical Sciences / 1043 Vol. 83, No. 7, Ju& 1994

Table 1-Percentage Entrapment of Suramln (0.07 mM) In Llposomes Prepared wlth Varlous Lipld Mlxtures

Lipids Amount (mM) Entrapmenta (%)

DPPC DPPCICHL DPPC DPPCICHL DPPCICHL DPPCICHLIST DPPCICHL/PG DPPC DPPC/CHL DPPCKHL DPPC DPPCICHL

3 3: 1.3 6 6:2.6 6 6 6:6:0.6 6:6: 1.2 12 125.1 12:12 25 25:25

25.9 f 1.8 8.2 f 0.8 37.0 f 1.4 8.8 f 2.5 2.3 f 0.3 30.3 f 7.1 2.5 f 0.4 50.6 f 2.7 9.1 f 2.7 4.3f 1.2 64.8 f 1.6 14.4 f 1.5

Table 2-Percentage Entrapment of Suramln (Varlous Concentratlons) In Llposomes Prepared wlth Varlous Concentratlons of DPPC

Amount EntrapmentB Amount Entrapmentd DPPC (mM) DPPC (mM) ( % I

0.14 mM Suramin 3 23.4 f 2.8 12 44.1 f 1.5 6 30.6 f 4.3 25 56.3 f 5.2

6 17.8 f 0.4

6 11.5 f 0.3

0.35 mM Suramin

0.70 mM Suramin

a Average of three independent measurements f SD.

Table 3-Percentage Entrapment of Sulfonated Compounds (0.07 mM) In Llposomes Prepared wlth Varlous Concentrations a Average of three independent measurements f SD.

of Llpld laser light scattering technique and may not be highly accurate. We estimate that the larger sized liposomes are 5-10 pm in size as estimated by the laser confocal microscope, which agrees with the laser light Lipids Amount (mM) EntrapmenP (%)

~

scattering data. Evans Blue Differential Scanning Calorimetry Study-Liposomes were DPPC 6 51.6 f 1.9

6:2.6 25.0 f 1.6 prepared by the same procedure described above. A Perkin-Elmer DSC-7 differential scanning calorimeter (DSC) was employed to evaluate possible suramin-phospholipid interactions. Phospholipid dispersions (15 rL) containing varying suramin concentrations were sealed in an DPPC 6 5.5 f 1.5

DPPC/CHL 1,5-NaphthalerIedisulfonic Acid, Disodium Salt

- .

3-Amino-2,7-Naphthalenedisulfonic Acid, Sodium Salt DPPC 6 1.2 f 0.3

a Average of three independent measurements f SD.

aluminum sample pan andmeasured against a reference pan containing normal saline (15 pL). Samples were scanned from 10 to 50 "C at 10 "C/min. The temperature accuracy of the instrument was calibrated using indium and cyclohexane standards.

Results Liposomal Entrapment of Suramin-Table 1 gives the

liposomal entrapment for suramin from aqueous solutions (5 mL) containing 0.07 mM of suramin. The liposome compositions studied were DPPC alone, DPPC/CHL (molar ratio 7:3 or l:l), DPPC/CHL/ST (molar ratio l O : l O l ) , and DPPC/CHL/PG (molar ratio 1010:2). The entrapment percentage is expressed as the percent suramin entrapped after removal of the free suramin by successive centrifugation and redispersion; the results are the average of three independent measurements. For DPPC liposomes, the entrapment percentage was 25.9% for 3 mM phospholipid. The entrapment pecentage increased as a function of increasing DPPC content, resulting in 37 % entrapment for 6 mM DPPC, 50.6% for 12 mM DPPC, and 64.8% for 25 mM DPPC. Incorporation of 30% or 50% cholesterol in the DPPC liposome decreased suramin entrapment. As shown in Table 1, the entrapment percentage was reduced by %fold to over 10- fold upon incorporation of cholesterol compared with the entrapment for the equivalent amount of DPPC alone. Incor- poration of positively charged stearlyamine (5 mol %) to DPPC/ CHL liposomes increased the entrapment from 2.3 % to 30.3%. The entrapment was not affected by the incorporation of negatively charged phosphatidylglycerol in DPPC/CHL lipo- somes.

Table 2 presents the liposomal entrapment employing aqueous solutions (5 mL) containing various amounts of suramin. When 0.14 mM of suramin was used, the entrapment percentage was lower than that with 0.07 mM but increased as a function of increasing DPPC (Tables 1 and 2). The entrapment decreased from 37% (Table 1) to 11.5% (Table 2) as suramin increased from 0.07 to 0.7 mM with DPPC at 6 mM.

Table 3 gives the liposomal entrapment for Evans blue, disodium 1,5-naphthalenedisulfonic acid, and sodium 3-amino- 2,7-naphthalenedisulfonic acid. The entrapment of Evans blue was 51.6% in DPPC liposomes for 6 mM phospholipid with 0.07 mM Evans blue, which was comparable to that for suramin. The

entrapment percentage, however, decreased by about one-half when Evans blue was incorporated into 7:3 (DPPC/CHL) liposomes. The entrapment of disodium 1,5-naphthalendisul- fonic acid (5.5 % and sodium 3-amino-2,7-naphthalenedisulfonic acid (1.2%) was low compared to that of suramin or Evans blue in DPPC liposomes.

Effect of Suramin on the phase-Transition Temperature of DPPC-Differential scanning calorimetry thermograms obtained with various amounts of suramin combined with DPPC (3 mM) are shown in Figure 1. In the absence of suramin, a pretransition peak at 38 "C was observed and the main transition occurred a t -42 "C. These results correspond to data reported previously for DPPC d i s p e r s i o n ~ . ~ ~ J ~ . ~ Suramin induced a decrease in both the main transition and pretransition tem- peratures of -2 "C when the molar ratio (suramin/DPPC) was 0.002. The pretransition peak disappeared a t a molar ratio of 0.05, while a t 0.1 a second peak appeared overlapping with the main transition endotherm. From these observations, it is apparent that an interaction occurred between suramin and DPPC. Similar changes in the DSC thermograms of DPPC dispersions have been shown with other interacting com- po~nds.~~-23

Discussion Suramin is a large polyanion (MW 1429.21) with six sulfonic

acid groups which are negatively charged under physiological conditions. Like other polyanionic substances, such as poly- acrylic acid, polyvinyl sulfate, and dextran sulfate, suramin has been shown to have a high binding affinity to a large number of proteins and en~ymes.6,~~,25 Such interactions of polyanions with phosphatidylcholine vesicles have not been well-studied. Evidence for interaction between suramin and DPPC was observed in the DSC thermograms which showed that suramin caused the disappearance of the pretransition peak, reduced the phase-transition temperature, and induced a second thermal

1044 /Journal of Phermeceutial Sciences Vol. 83, No. 7, July 1994

\ Ratio=O.l

I\ I 1 Rati0=0.002

I I ~ I ~ - ~ 30.0 40.0 l . 0

Temperature (“C) Flguro 1-Differential scanning calorimetry thermograms of DPPC dispersions in sodium suramin solutions. The suraminlDPPC mole ratio is indicated on each thermogram.

transition peak of DPPC dispersions (Figure 1). We postulate that this effect depends on the amount of drug bound to the liposomal membrane or intercalated into the bilayer.

Suramin entrapment increased as a function of increasing DPPC (Table 1). A similar trend in entrapment with increasing DPPC was observed with either 0.07 or 0.14 mM of suramin (Tables 1 and 2). However, the entrapment percentage decreased with increasing amounts of suramin for a constant amount of phospholipid (Tables 1 and 2). This behavior suggests that suraniin entrapment is saturable and different from that expected for noninteracting hydrophilic compounds wherein a constant fraction of the aqueous drug solution would be entrapped.

Evims blue was also highly entrapped in DPPC liposomes. Evan!, blue is structurally similar to suramin in that it has four sulfonate groups distributed over a large molecule. The lower entralpment of disodium 1,5-naphthalenedisulfonic acid and sodium 3-amino-2,7-naphthalenesulfonic acid indicate that smaller and less sulfonated anions interact less strongly with DPPC.

Suramin entrapment would not be as high as that observed if it were only entrapped in the internal aqueous compartments of the liposomes. Gruner et al. reported that MLVs prepared with 100 mg of egg PC entrapped only 2 76 of radiolabeled inulin and 2% of 22NaC1.26 Such entrapment is typical of hydrophilic compounds which do not interact with phospholipid. In addition to high entrapment, we have found that the suramin concentra- tion in the supernatant solution drawn from the first centrifuged sample was 2-%fold less than the originalsuramin concentration. From these observations, we assume that a major portion of the entrapped suramin results from binding onto the surface of or intercalation into the liposomal bilayer. The binding or intercalation may be attributed to the molecular size and the presence of polysulfonate groups in suramin or Evans blue.

Since liposomes prepared by this thin-film hydration method are multilamellar, we propose that suramin is distributed among the internal bilayers by binding, intercalation, or encapsulation. Even though the entrapment mechanism is unknown, little suramin would be bound to the outer lipsomal surface. Thus, <lo % of the entrapped suramin would be expected to dissociate rapidly in the presence of serum or upon dilution in the blood.

Much less suramin is entrapped when 30-50 mol % cholesterol is added to DPPC liposomes (Table 1). Also, Evans blue entrapment decreased by about one-half when equivalent cholesterol was added to DPPC liposomes (Table 3). Cholesterol condenses phospholipid bilayers which may make the DPPC less accessible to interaction with suramin. Such behavior has been proposed by Gross and Ehrenberg, who observed that increasing the cholesterol content in phosphatidylcholine liposomes caused a decrease in binding constant of both a hematoporphyrin derivative and photofrin II.fl,2e They obtained lower binding constants to egg PC/CHL liposomes compared to egg PC liposomes for these dyes. Such behavior for suramin suggests that it ionically interacts with the cationic choline moieties thereby bridging two DPPC molecules in the bilayer with intercalation of the more hydrophobic middle of the suramin molecule into the bilayer. The addition of cholesterol would interrupt such a bridging/intercalation mechanism. The low entrapment of the smaller charged molecules (1 ,&naphthalene- disulfonic acid and 3-amino-2,7-naphthalenedisulfonic acid) supports the bridging mechanism. They are too small to ionically bridge two DPPC molecules and are not sufficiently hydrophobic to intercalate into the bilayer. Thus, they show entrapment which involves only material dissolved in the entrapped aqueous phase.

The suramin entrapment increased significantly with the addition of positively-charged stearylamine (5 mol % ) to DPPC/ CHL liposomes compared to that of neutral DPPC/CHL liposomes. Interaction between negatively-charged suramin and positively-charged alkylamine in the liposomal membrane would also be expected to increase entrapment by ionic interaction. Such behavior has been shown by Kano and Fendler, who studied the liposomal entrapment of pyranine (trisodium 8-hydroxy- 1,3,6-pyrenetrisulfonate), which is highly anionic and completely ionized over a wide pH range.29 The anionic pyranine was repelled from the surface of the negatively-charged liposomes and was attracted to the positively-charged inner and outer surfaces of cationic liposomes. They found pyranine was highly incorporated (87 76 ) into positively-charged (DPPCICHL) liposomes containing stearylamine. Suramin’s high entrapment in DPPCISTICHL liposomes could also be explained by such an ionic interaction. As expected, if such interactions are primarily ionic, the addition of negatively-charged PG had no effect on suramin entrapment.

The high liposomal entrapment of suramin may have ap- plication in reducing its toxicity or increasing its efficacy. Liposomal suramin may be better delivered to solid tumors or to cell surface binding sites for various growth factors. In AIDS, delivery of liposomal suramin to HIV-infected lymphocytes may better suppress reverse transcriptase activity and decrease the dose required for effective therapy. Therefore, it is expected that suramin’s therapeutic index may be substantially improved with incorporation into liposomes.

Further investigations are underway to better elucidate the mechanism of interaction of suramin with zwitterionic phos- pholipid vesicles. It is possible that the entrapment and/or targeting of suramin may be further improved with other phospholipid compositions or different methods of liposome preparation. Also, other polyanionic cornpounds like dextran sulfate and pentosan polysulfate with antiviral or other phar- macological activity might be more efficacious when similarly incorporated into liposomes.

Journal of PharmaceutiCel Sciences / 1045 Vol. 83, No. 7, July 1994

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