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Evaluation of Blood Clearance Rates and Biodistribution of Poly(2-oxazoline)-Grafted Liposomes§
Evaluation of Blood Clearance Rates and Biodistribution of Poly(2-oxazoline)-Grafted Liposomes§ SAMUEL ZALIPSKY†X, CHRISTIAN B. HANSEN‡, JAN M. OAKS†,
AND
THERESA M. ALLEN‡
Received September 27, 1995, from the †SEQUUS Pharmaceuticals, Inc., 960 Hamilton Court, Menlo Park, CA 94025, and ‡Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2H7, Canada. Final revised manuscript received November 16, 1995. Accepted for publication November 20, 1995X. Abstract 0 Two amphipatic polymers of the poly(2-oxazoline) family, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ), were synthesized with the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently linked to the carboxyl groups of the polymers, resulting in conjugates which incorporate readily into liposomes. Systematic evaluation of plasma clearance kinetics and biodistribution of liposomes containing hydrogenated soy phosphatidylcholine, cholesterol, and 5 mol % the polymer−lipid conjugates in mice revealed the following. Both polymers, PMOZ and PEOZ, exhibited long plasma lifetimes and low hepatosplenic uptake. PMOZ was more effective at decreasing blood clearance rates than PEOZ. The best results, which were quantitatively comparable to the results obtained with the optimized preparations of methoxypolyethylene glycol(PEG)-2000-grafted liposomes, were obtained with formulations containing PMOZ of molecular weight 3260.
Introduction To overcome some of the limitations of liposomes as drug carriers considerable effort has been directed toward extending their plasma lifetimes and diminishing their accumulation in the organs of the mononuclear phagocyte system (MPS, also known as the reticuloendothelial system) (reviewed in ref 1). Since the current understanding of the plasma clearance mechanisms involves interaction of lipid vesicles with plasma proteins,2 modification of liposomal surfaces with the goal to minimize such interactions was one of the main directions of this effort.3,4 Heretofore the best results were achieved by incorporation into liposomal formulations of 3-10 mol % of methoxypolyethylene glycol-2000-distearoylphosphatidylethanolamine (mPEG-DSPE) conjugates.5-8 This results in lipid vesicles with PEG-grafted surfaces. The liposomes of this type exhibit dose-independent pharmacokinetics accompanied with remarkable persistence in vivo.3,9 For example, in humans plasma half-lives of approximately 48 h with enhanced malignant tissue accumulation were observed for PEGylated liposomes.10 The ability of PEG to elicit this effect, when grafted onto the surface of liposomes, has been explained by its chains’ high mobility associated with conformational flexibility and water-binding ability.6,11-13 These properties all contribute to the so-called steric stabilization effect, which results in the well-known propensity of PEG to exclude proteins, other macromolecules, and particulates from its surroundings. It is reasonable to assume that other polymers might possess similar properties and thus, when § Abbreviations: MPS, mononuclear phagocyte system ; PL, phospholipid; POZ, poly(2-oxazoline); PMOZ, poly(2-methyl-2-oxazoline); PEOZ, poly(2-ethyl-2-oxazoline); PEG, polyethylene glycol; mPEG, methoxy-PEG; DSPE, distearoylphosphatidylethanolamine; HSPC, hydrogenated soy phosphatidylcholine; CHOL, cholesterol; DP, dgree of polymerization; TI, tyraminylinulin, GPC, gel permeation chromatography, DMF, N,N′dimethylformamide. X Abstract published in Advance ACS Abstracts, January 1, 1996.
© 1996, American Chemical Society and American Pharmaceutical Association
grafted onto liposomal surfaces, they too should be able to exert similar influences on the vesicles. One class of polymers, poly(2-oxazolines) (POZ), impressed us as particularly promising.14 Similarly to PEG, the backbone of poly(2-oxazoline) is composed of carbon-carbonheteroatom repeating units (see Scheme 1). This suggests similar segmental flexibility of the polymers in solution, which is thought to be of importance for exerting the beneficial “PEG properties” mentioned above.13 Because the pendant N-linked acyl residue is important in influencing the physical properties of POZ, the choice of these groups was limited to the two residues which confer amphipatic properties to these polymers, namely the acetyl and propionyl groups (R ) Me, Et). Indeed, after examination of solubility properties we found a great deal of similarity between PEG and poly(2-methyl-2oxazoline) (PMOZ) and particularly poly(2-ethyl-oxazoline) (PEOZ).15 These similarities in both structure and properties prompted our examination of the two amphipatic polyoxazolines, PMOZ and PEOZ, as modifiers of the liposomal surface. Our preliminary experiments with the POZs showed that both polymers are effective in prolonging liposome circulation time in rats.15 Here we are presenting the results of a systematic evaluation of the plasma clearance kinetics and biodistribution of POZ-grafted liposomes in mice.
Experimental Procedures GeneralsPotassium iodide and methyl p-toluenesulfonate were from Fluka (Ronkonkoma, NY). Ethyl 3-bromopropionate, 2-methyl2-oxazoline, 2-ethyl-2-oxazoline, glutaric anhydride, anhydrous acetonitrile, and chloroform were purchased from Aldrich (St. Louis, MO). Both oxazoline monomers were distilled prior to polymerization over NaOH. A sample of poly(2-ethyl-2-oxazoline) of nominal molecular weight 5000 (Aquazol-5) was obtained from Polymer Chemistry Innovations, Inc. (State Collage, PA). Gel permeation chromatography (GPC) was measured using a Schimadzu-10A instrument equipped with a PL-Gel 5 µm, 1000 Å column (Polymer Laboratories, Inc., Amherst, MA) calibrated with PEG standards in DMF at 40 °C. The data was processed using a GPC program on a Chromatopac C-R7A. 1H-NMR spectra were measured in CDCl or CD OD at 360 MHz on 3 3 a GE instrument by Acorn NMR (Fremont, CA). Hydrogenated soy phosphatidylcholine (HSPC) was obtained from SEQUUS Pharmaceuticals Inc. (Menlo Park, CA). The synthesis of mPEG2000-DSPE was previously described in detail.5,8,16 Cholesterol (CHOL) was purchased from Avanti Polar Lipids (Birmingham, AL). Na125I was purchased from Amersham (Oakville, Canada). Tyraminylinulin was synthesized and 125I-labeled (125I-TI) as previously described.17 Preparation of End-Group-Carboxylated POZssPolymerization reactions were conducted in screw-cap tubes dried at 110 °C, for 24 h prior to their use. The tubes were charged with freshly distilled monomer (5 mL), dry acetonitrile (7 mL) and initiator, purged with nitrogen, sealed, and left stirring in an oil bath at 80 °C for 18 h. Freshly distilled methyl tosylate or alternatively ethyl 3-bromopropionate with 1.1 equiv of KI was used as initiators. Since the 2-oxazoline polymerization process is not self-terminating, also known as living polymerization, the amount of initiator (mole ratio of initiator to monomer) was determined by the desired degree of polymerization (DP). Termination was performed by two equally effective procedures converting the terminal oxazolinium ions into N-(2-hydroxyethyl)
0022-3549/96/3185-0133$12.00/0
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Scheme 1sSynthesis of polyoxazoline−DSPE conjugates. An asterisk denotes termination by either one of the two methods described in the experimental section. Abbreviations: DCC, dicyclohexylcarbodiimide; HOSu, N-hydroxysuccinimide; Int-X, initiator; X, halide or sulfonate leaving-group residue; TEA, triethyl-amine; -OTs, tosylate. groups. The first method adapted from Kobayashi et al.18 involved addition of solid sodium carbonate and water and heating the reaction mixture at 80 °C for 6 h. Alternatively, a methanolic solution of KOH (0.5 M) was added to the polymerization mixture followed by stirring at 25 °C for 2 h according to the procedure of Riffle and co-workers.19 When ethyl 3-bromopropionate was used as initiator, addition of 2.5 equiv of KOH caused both termination and formation of a carboxyl group by hydrolysis of the ethyl ester at the initiation end of the polymer. Purification of the polymers was accomplished by water dilution of the terminated mixtures to 50 mL and dialysis against 50 mM NaCl, 2% acetic acid, and water (3 × 3 L each) using 3500 molecular weight cutoff Specrta/Por dialysis membrane (Spectrum, Los Angeles, CA). The content of the dialysis bag was lyophilized and then further dried in vacuo over P2O5. Typical recoveries were 70-90%. Polymers initiated with methyl tosylate (including Aquazol-5) were converted into their carboxylated derivatives by reaction with glutaric anhydride. For this purpose POZ was azeotropically dried in benzene (in the case of PEOZ) or acetonitrile (for PMOZ) at a ratio of 10 mL of solvent/g of polymer, concentrated to approximately one-fourth of the initial volume, treated with a 5-fold excess of the anhydride and a 10-fold excess of pyridine, and refluxed for 4 h. The reaction mixtures were evaporated to dryness and dissolved in water and the products recovered by the dialysis, lyophilization, and drying as described above. The polymers were further characterized according to the well-established protocols.18-20 Preparation of POZ-DSPE ConjugatessCarboxylated POZ (0.3 mmol of either glutarate or propionate end groups) was dissolved in chloroform (10 mL) and treated with N-hydroxysuccinimide (0.4 mmol) and dicyclohexylcarbodiimide (0.4 mmol). The solution was stirred overnight at 25 °C and then filtered from precipitated dicyclohexylurea. DSPE (0.25 mmol) was added to the solution followed by triethylamine (2.5 mmol). The reaction mixture was gently warmed up to 40-45 °C for 5-10 min while being stirred. During this time a clear solution was formed as all the DSPE reacted
134 / Journal of Pharmaceutical Sciences Vol. 85, No. 2, February 1996
(confirmed by TLC with ninhydrin staining). The solution was neutralized with acetic acid (2.5 mmol) and evaporated to dryness. The product was purified of unreacted polymer and lower molecular weight reactants using dialysis through 300 000 molecular weight cutoff Spectra/Por CE membrane and recovered as previously described for the PEG-DSPE conjugates.16 The following TLC (silica gel G, visualized with I2 vapor) eluents were used to check the purities of the conjugates: n-propanol/30% aqueous ammonia (7:3), CHCl3/ ethanol/30% aqueous ammonia (3:7:2), CHCl3/ethanol/H2O (80:18:2 and 130:70:8). In the last two solvent mixtures, PMOZ and its conjugates were stationary while PEOZ and its conjugates, as well as free DSPE, were readily separated and detected. The structure of the conjugates was confirmed by 1H-NMR spectra, exhibiting the characteristic peaks of both lipid and polymer components: δ (ppm) 0.9, 1.26, 1.58, 2.28, minor peaks 4-4.5, and 5.2 for DSPE; 2.1 and 3.5 for PMOZ; and 1.13, 2.3-2.4, and 3.5 for PEOZ. The broad polymer peaks often overlapped with minor absorbances, influencing adversely our ability to accurately integrate the spectra. Preperation of LiposomessLiposomes were composed of HSPC: CHOL:mPEG-DSPE (2:1:0.1 molar ratio) or HSPC:CHOL with various amounts of either PEOZ-DSPE or PMOZ-DSPE of various polymer chain lengths. Liposomes were prepared by hydrating dry lipid films in 25 mM HEPES, 140 mM NaCl, pH 7.4 buffer, with the aqueous space label 125I-TI at a lipid concentration of 10 mM. The resulting multivesicular preparations were then passed through 0.10.08 µm polycarbonate membranes (Nuclepore Corp., Pleasanton, CA) using a Lipex extruder (Lipex Biomembranes, Vancouver, B. C.), to give primarily unilamellar vesicles with diameters of 99-112 nm.21,22 The size of the liposomes were determined by dynamic light scattering using a Brookhaven BI90 particle sizer (Brookhaven Instruments, Holtsville, NY). Free 125I-TI was separated from the liposomes by chromatography over a Sepharose CL-4B column with 25 mM HEPES, 140 mM NaCl, pH 7.4 buffer. In Vivo ExperimentssOutbred female CD1(ICR)BR mice were purchased from Charles River Canada (St. Constant, Canada) and maintained in standard housing. Mice (three per group) were given a single bolus iv injection via the tail vein of 0.2 mL of liposomes (0.5 µmol of PL/mouse) containing 2-4 × 105 cpm of 125I-TI. At different times postinjection, animals were anaesthetized with halothane and sacrificed by cervical dislocation. Major organs (liver, spleen, lung, heart, and kidney), blood (100 µL), thyroid, and carcass (remainder of the animal) were collected and counted for 125I label in a Beckman 8000 γ counter. Blood correction factors23 were applied to all samples. The data is expressed as the percentage of counts in each organ relative to the total counts remaining in vivo at each time point.24 Pharmacokinetic parameters of the liposomes were calculated using the polyexponential curve stripping and least squares parameter estimation program RSTRIP (Micromath, Salt Lake City, UT).
Results and Discussion Polyoxazolines are usually synthesized by cationic ringopening polymerization initiated by a variety of alkylating agents and terminated by nucleophiles.14 The nature of the polymerization process allows the use of termination or initiation ends of the polymer chains for placement of functionalized residues for subsequent conjugation with lipids. Using both termination and initiation-based approaches for polyoxazoline functionalization, we prepared several carboxylterminated POZs, as depicted in Scheme 1. Relying on wellestablished literature procedures,18-20 our initial experiments utilized methyl p-toluenesulfonate-initiated polymerization of 2-oxazolines followed by a reaction of hydroxy-terminated polymer with glutaric anhydride for introduction of carboxylic acid group.15 Since both POZs are very hygroscopic and pick up moisture during their recoveries by precipitation, often resulting in pasty solids, we used dialysis followed by lyophilization and drying in vacuo for purification and isolation of polymer derivatives from both polymerization and the following derivatization reactions. Although very simple, this method of POZ isolation is quite time-consuming. In order to minimize the number of manipulations, we developed an alternative approach to preparation of end-carboxylated POZ.
Table 1s Summary of the Polymers and Their DSPE Conjugates Polymer Type
Mn [Mw/Mn]
DPa
Conjugate Type (see Scheme 1)b
MW of DSPE Conjugate
PEOZ
1690 [1.15] 3070 [1.44] 4660 [1.56]c 4420 [1.18] 2020 [1.60] 2290 [1.12] 3260 [1.53] 4330 [1.18] 2000
17 31 47 45 24 27 38 51 45
P P G P G P P P U
2440 3820 5410 5170 2770 3040 4010 5080 2750
PMOZ
mPEG a
Degree of polymerization (DP) was calculated by dividing Mn by the molecular weight of single repeating unit (85 for PMOZ, and 99 for PEOZ). b P ) propionate; G ) glutarate; U ) urethane. c Derivative of Aquazol-5.
Figure 1sThe effect of the type of linkage between the poly(2-oxazoline) polymer and DSPE on the blood elimination of liposomes. (A) Poly(2-ethyl-2-oxazoline) and (B) poly(2-methyl-2-oxazoline) were conjugated to DSPE by either a propionate (P) or glutarate (G) linkage (see Scheme 1). Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (A) (b) PEOZ4420-P−DSPE and (9) PEOZ4660-G−DSPE; (B) (9) PMOZ2020-G−DSPE and (b) PMOZ2290-P−DSPE.
It relied on the use of ethyl 3-bromopropionate as the initiator for 2-oxazoline polymerization. The polymerization reactions proceeded in presence of a 10% molar excess of potassium iodide in dry acetonitrile and resulted in polymers of similar yields and dispersities as in methyl tosylate-initiated reactions. Upon termination of the polymerization process with either aqueous carbonate or methanolic potassium hydroxide, the ethyl propionate end group was converted into a free carboxyl residue. The molecular weights of the synthesized polymers were in the range Mn 1500-5000 Da with low dispersity (Mw/Mn 1.1-1.6), as determined by GPC.25 The end-carboxylated polyoxazolines, prepared by both functionalization approaches shown in Scheme 1, were converted in situ into their succinimidyl esters and then coupled to amino group of DSPE in chloroform under conditions previously used for preparation of PEG-lipids.26 We characterized the products by TLC and 1H-NMR spectra. Some properties of the polyoxazolines and their conjugates are summarized in Table 1. Liposomes containing the POZ conjugates listed in Table 1 were prepared (105 ( 6 nm) with the standard composition HSPC/CHOL/POZ-DSPE 2:1:0.1. Thus the polymer-lipid conjugate was always present at 5 mol % of phospholipid content. The POZ-grafted liposomal preparations were labeled with 125I-tyraminylinulin and then injected into mice for determination of their longevity in circulation and their biodistribution parameters, according to previously described methodologies.5,27 As can be seen from Figure 1, polymers of the same (or similar) molecular weights grafted onto liposomes through either P- or G-type of linkages positioned at either initiation or termination ends, respectively, produce approximately the same plasma clearance rates. Although aliphatic ester-containing conjugates similar to G-type POZDSPE are known to be labile in aqueous buffers,28 the
hydrolysis rates are usually too slow to have an effect on the clearance rates.29 Since the results obtained with either Por G-type of lipid conjugates were essentially the same and since the P-type of conjugates was more convenient to prepare, we continued our experimentation with P-type of conjugates only. Figure 2 illustrates the relationship between circulation times and molecular weights of the grafted polymers. Among the PEOZ-containing formulations, variation of molecular weight of the grafted polymers produced hardly any changes in the longevity of the liposomes in blood circulation (Figure 2A). Preparations containing PMOZ-3260 showed the best results among PMOZ liposomes (Figure 2B), with PMOZ-4330grafted vesicles producing only slightly inferior results. Comparison of the best-performing PMOZ liposomes with preparations containing mPEG-2000, previously found to be optimal among PEG-grafted liposomes,5,8 and with PEOZ4420-containing liposomes is shown in Figure 3. Although, as shown in Figure 2A, there was no significant difference between the tested PEOZ preparations, PEOZ-4420 was chosen as “the best” based on slightly higher blood levels of its liposomes at later time points (24 and 48 h). The comparison of the three preparations (Figure 3) demonstrates that lipid vesicles grafted with PMOZ-3260 produced a similar pharmacokinetic curve to liposomes containing mPEG-2000. While the results obtained with PEOZ-4420-modified vesicles were not quite as favorable, this preparation still can be viewed as long-circulating in comparison to “classical” (nonPEGylated) liposome formulations. The biodistribution results summarized in Table 2 showed good correlation between longevity in circulation and low uptake by liver and spleen. The biodistribution results also confirm the similarity between the two longest circulating formulations, containing mPEG2000 or PMOZ-3260. The pharmacokinetic parameters of the polymer-grafted liposomes and their “classical” counterparts (i.e., not containing polymer) are given in Table 3. “Classical” liposomes were either neutral or contained 5 mol % of phosphatidylglycerol (PG) to provide the same amount of negative charge carried by the polymer-grafted liposomes. The mean residence time, half-life, and the area under concentration-time curve (AUC) are significantly higher for the polymercontaining liposomes compared to liposomes lacking polymer. The elimination rate constant is significantly lower for the polymer-containing liposomes. Note that the degree of polymerization (DP) values of the best performing polymers, mPEG-2000, PMOZ-3260, and PMOZ-4330, are in 38-51 range (Table 1). This might be important since DP determines the number of “bending units” and as such is directly related to the degree of conformational freedom of polymer chains. The latter parameter is thought to be consequential to the beneficial influence of the polymers on liposome blood clearance.6,11-13,15 The observation that the higher molecular weight PMOZ conjugate was cleared from
Journal of Pharmaceutical Sciences / 135 Vol. 85, No. 2, February 1996
Table 2sTissue Distributions of Polymer-Grafted Liposomes in Micea Polymer
Blood
Liver
mPEG-2000 PMOZ-3260 PEOZ-4420
77.3 ± 1.1 82.2 ± 3.0 61.3 ± 6.7
11.8 ± 0.9 10.7 ± 1.9 21.1 ± 3.0
mPEG-2000 PMOZ-3260 PEOZ-4420
27.2 ± 8.3 22.1 ± 4.0 9.7 ± 3.0
22.2 ± 1.0 32.1 ± 1.0 44.7 ± 5.8
Spleen 2 h postdose 1.7 ± 0.6 1.3 ± 0.4 5.3 ± 1.0 24 h postdose 4.8 ± 0.5 3.8 ± 0.7 9.2 ± 2.2
Lung
Heart
Kidney
Carcass
0.6 ± 0.4 0.3 ± 0.3 0.3 ± 0.1
0.5 ± 0.1 0.8 ± 0.1 0.4 ± 0.1
1.7 ± 0.8 1.9 ± 0.3 1.5 ± 0.5
6.5 ± 0.5 2.8 ± 3.7 10.0 ± 5.2
0.2 ± 0.0 0.5 ± 0.1 0.3 ± 0.1
0.6 ± 0.2 0.4 ± 0.1 0.3 ± 0.1
3.2 ± 0.4 3.5 ± 0.7 2.2 ± 0.4
41.5 ± 8.8 36.9 ± 2.0 32.5 ± 4.9
a Liposomes (ca. 100 nm) were composed of HSPC:CHOL (2:1, molar ratio) containing 5 mol of either mPEG-2000−DSPE (n ) 3), PMOZ-3260−DSPE (n ) 3), or PEOZ-4420−DSPE (n ) 9). Both polyoxazoline compounds contained the proprionate linkage. Tissue distributions were determined for 125I-tyraminylinulin-labeled liposomes (0.5 µmol of phospholipid per mouse) at 2 and 24 hours after iv injection. Mean ± SD.
Figure 3sComparison between polyethylene glycol and the polyoxazolines on elimination of liposomes from blood. Liposomes (ca. 100 nm) were composed of HSPC:CHOL, 2:1 molar ratio with 5 mol % polymer−lipid. Both POZ were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (9) mPEG2000−DSPE, (b) PMOZ3260-P−DSPE, and (2) PEOZ4420-P−DSPE. Table 3sPharmacokinetic Parametersa
Figure 2sThe effect of polymer molecular weight of (A) poly(2-ethyl-2-oxazoline)− DSPE and (B) poly(2-methyl-2-oxazoline)−DSPE on blood elimination of liposomes. Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Polymer lipids were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (A) (2) PEOZ4420P−DSPE, (1) PEOZ3070-P−DSPE, and (9) PEOZ1690-P−DSPE; (B) ([) PMOZ4330P−DSPE, (b) PMOZ3260-P−DSPE, and (9) PMOZ2290-P−DSPE.
blood circulation slightly faster than the PMOZ-3260 analog might be explained by the known propensity of lipid-linked polymer chains of increased length to dissociate faster from lipid bilayers than their shorter chain counterparts.30 A similar rationale was used to explain the differences between mPEG-2000- and mPEG-5000-grafted liposomes.29 Since, the rate of dissociation and thus bilayer retention of polymerlipids has also been shown to be hydrophobic-anchor-dependent, we chose to prepare the POZ conjugates from DSPE, previously shown to be the optimal lipid anchor.31 Finally, we looked at the relationship between the plasma persistence of formulations and concentration of POZ-lipid. As can be seen from Figure 4, at 24 h postinjection variation of the mol percentage of either polymer conjugate had a noticeable effect. For PEOZ-4420-grafted liposomes, increasing the polymer content from 2 to 5 mol % resulted in a more than doubling of the blood levels, with no further increase at higher mol percentages of the conjugates. PMOZ-containing liposomes also exhibited consistently higher blood levels with 136 / Journal of Pharmaceutical Sciences Vol. 85, No. 2, February 1996
Liposome Composition
MRTb (h)
AUC0-∞c (nmol h/mL)
k10d (h-1)
t1/2R (h)
t1/2β (h)
HSPC:CHOL:mPEG2000−DSPE HSPC:CHOL:PMOZ3260-P−DSPE HSPC:CHOL:PEOZ4420-P−DSPE HSPC:CHOL HSPC:CHOL:PG
23.5 21.7 13.8 9.0 5.3
4761 4388 2267 2031 1271
0.06 0.06 0.10 0.15 0.24
3.16 5.1 0.77 0.01 0.05
17.7 17.8 9.9 6.2 3.7
a Mice were given a single bolus iv injection, via the tail vein, with liposomes (0.5 µmol of phospholipid, ca. 100 nm in diameter). Liposomes were composed of HSPC:CHOL at a 2:1 molar ratio with PG, POZ−DSPE or mPEG−DSPE present at 5 mol % of phospholipid. Blood samples were taken at various times up to 48 h postinjection. All pharmacokinetic parameters were calculated from a twocompartment open model (biexponential curve fit) using the program RSTRIP (Micromath, Salt Lake City, UT). b Mean residence time. c Area under the blood concentration vs time curve. d Elimination rate constant from the central compartment.
increasing content of polymer-lipid in the formulations. At all polymer concentrations, PMOZ-containing liposomes yielded higher blood levels than their more hydrophobic PEOZ counterparts. Thus the difference between the effects of PMOZ or PEOZ on liposome circulation times became more pronounced with increasing mol percentage of polymer-lipid components in the formulations, until the effect reached saturation between 5-10 mol %. It is pertinent to note that during early attempts to prepare long-circulating liposomes it was observed that GM1-containing formulations32 exhibited circulation longevity in mice but not in rats.33 Although the mechanism of liposomal surface protection by grafted synthetic polymers, like POZs, must be different from that of GM1, it was important to confirm that polyoxazoline-grafted liposomes exhibited plasma longevity
Figure 4sThe effect of increasing the poly(2-oxazoline) content in liposomes on blood elimination at 24 hours postinjection. Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Polymer lipids were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per methods. Key: (b) PMOZ3260-P−DSPE and (2) PEOZ4420-P−DSPE.
and low hepatosplenic uptake in more than one species. From the experimental observations reported here and in our earlier communication,15 it appears that POZs convey long circulation properties to liposomes in both rats and mice, similar to PEG. Quantitatively the effect is also comparable to that of PEG. Although there have been other attempts to utilize synthetic polymers other than PEG for preparation of long-circulating liposomes,34-36 to the best of our knowledge to date only PMOZ liposomes in mice and both PMOZ and PEOZ liposomes in rats exhibited the same ability to avoid MPS uptake and to extend blood lifetimes as their mPEG-containing counterparts. Although there is a considerable amount of literature on POZs14 thus far only a few papers dealt with applications of these polymers to biomedical problems.20,37-40 These include the evaluation of PMOZ as a promising drug carrier.37 Our observations that at 48 h postdose the mice showed no signs of toxicity, abnormal behavior, or organ abnormalities are in agreement with the previously published observations suggesting the low toxicity and good biocompatibility of POZs.
Conclusions Our results demonstrate that two amphipatic polymers of the poly(2-oxazoline) family, PEOZ and PMOZ, are efficient in conveying long circulation properties to liposomes in mice. This finding, combined with the previously reported ability of these polymers to cause MPS evasion and longevity in circulation in rats, suggests that, similar to PEG, the effect maybe species-independent. Judging by the plasma clearance kinetics and biodistribution in mice, PMOZ-3260-grafted liposomes perform as well as mPEG-2000 liposomes, while PEOZ-containing preparations are somewhat inferior. To our knowledge, poly(2-methyl-2-oxazolines) linked to DSPE are the only polymers conjugates able to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as PEG. In light of these encouraging initial results, it is prudent to verify whether the usefulness of the amphipatic POZs also extends to other in vivo applications: conjugates of proteins, biocompatible surfaces, etc.
6. Blume, G.; Cevc, G. Biochim. Biophys. Acta 1993, 1146, 157168. 7. Klibanov, A. L.; Maruyama, K.; Beckerleg, A. M.; Torchilin, V. P.; Huang, L. Biochim. Biophys. Acta 1991, 1062, 142-148. 8. Woodle, M. C.; Matthay, K. K.; Newman, M. S.; Hidayat, J. E.; Collins, L. R.; Redemann, C.; Martin, F. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1992, 1105, 193-200. 9. Woodle, M. C.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1113, 171-199. 10. Gabizon, A.; Catane, R.; Uziely, B.; Kaufman, B.; Safra, T.; Cohen, R.; Martin, F.; Huang, A.; Barenholz, Y. Cancer Res. 1994, 54, 987-992. 11. Needham, D.; McIntosh, T. J.; Lasic, D. D. Biochim. Biophys. Acta 1992, 1108, 40-48. 12. Lasic, D. D.; Martin, F. J.; Gabizon, A.; Huang, S. K.; Papahadjopoulos, D. Biochim. Biophys. Acta 1991, 1070, 187-192. 13. Torchilin, V. P.; Papisov, M. I. J. Liposome Res. 1994, 4, 725739. 14. Kobayashi, S. Prog. Polym. Sci. 1990, 15, 751-823. 15. Woodle, M. C.; Engbers, C. M.; Zalipsky, S. Bioconjugate Chem. 1994, 5, 493-496. 16. Zalipsky, S. Bioconjugate Chem. 1993, 4, 296-299. 17. Sommerman, E. F.; Pritchard, P. H.; Cullis, P. R. Biochem. Biophys. Res. Commun. 1984, 122, 319-324. 18. Kobayashi, S.; Masuda, E.; Shoda, S.; Shimano, Y. Macromolecules 1989, 22, 2878-2884. 19. Sinai-Zingde, G.; Verma, A.; Liu, Q.; Brink, A.; Bronk, J. M.; Mirand, H.; McGrath, J. E.; Riffle, J. S. Macromol. Chem. Macromol. Symp. 1991, 42/43, 329-343. 20. Myamoto, M.; Naka, K.; Shiozaki, M.; Chujo, Y.; Saegusa, T. Macromolecules 1990, 23, 3201-3205. 21. Olson, F.; Hunt, C. A.; Szoka, F. C.; Vail, W. J.; Papahadjopoulos, D. Biochim. Biophys. Acta 1979, 557, 9-23. 22. Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. 23. Allen, T. M. In Liposomes in the Therapy of Infectious Diseases and Cancer; Lopez-Berestein, G., Fidler, I., Eds.; Alan R. Liss, Inc.: New York, 1989; Vol. 89; pp 405-415. 24. Allen, T. M.; Hansen, C. B.; Lopez de Menezes, D. E. Adv. Drug. Delivery Rev. 1995, 16, 267-284. 25. Nathan, A.; Zalipsky, S.; Erthel, S. I.; Agathos, S. N.; Yarmush, M. L.; Kohn, J. Bioconjugate Chem. 1993, 4, 54-62. 26. Zalipsky, S. In Stealth Liposomes; D. Lasic and F. Martin, Ed.; CRC Press: Boca Raton, FL, 1995; pp 93-102. 27. Allen, T. M.; Brandeis, E.; Hansen, C. B.; Kao, G. Y.; Zalipsky, S. Biochim. Biophys. Acta 1995, 1237, 99-108. 28. Zalipsky, S.; Seltzer, R.; Menon-Rudolph, S. Biotechnol. Appl. Biochem. 1992, 15, 100-114. 29. Zalipsky, S. Adv. Drug Delivery Rev. 1995, 16, 157-182. 30. Silvius, J. R.; Zuckermann, M. J. Biochemistry 1993, 32, 31533161. 31. Parr, M. J.; Ansell, S. M.; Choi, L. S.; Cullis, P. R. Biochim. Biophys. Acta 1994, 1195, 21-30. 32. Allen, T. M.; Chonn, A. FEBS Lett. 1987, 223, 42-46. 33. Yamauchi, H.; Yano, T.; Kato, T.; Tanaka, I.; Nakabayashi, S.; Higashi, K.; Miyoshi, S.; Yamada, H. Int. J. Pharm. 1995, 113, 141-148. 34. Torchilin, V. P.; Shtilman, M. I.; Trubetskoy, V. S.; Whiteman, K.; Milstein, A. M. Biochim. Biophys. Acta 1994, 1195, 181184. 35. Maruyama, K.; Okuizumi, S.; Ishida, O.; Yamauchi, H.; Kikuchi, H.; Iwatsuru, M. Int. J. Pharm. 1994, 111, 103-107. 36. Torchilin, V. P.; Trubetskoy, V. S.; Whiteman, K. R.; Caliceti, P.; Ferruti, P.; Veronese, F. M. J. Pharm. Sci. 1995, 84, 10491053. 37. Goddard, P.; Hutchinson, L. E.; Brown, J.; Brookman, L. J. J. Controlled Release 1989, 10, 5-16. 38. Shenouda, L. S.; Adams, K. A.; Zoglio, M. A. Int. J. Pharm. 1990, 61, 127-134. 39. Desai, N. P.; Hubbell, J. A. Biomaterials 1991, 24, 144-153. 40. Velander, W. H.; Madurawe, R. D.; Subramanian, A.; Kumar, G.; Sinai-Zingde, G.; Riffle, J. S. Biotechnol. Bioeng. 1992, 39, 1024-1030.
References and Notes 1. Lasic, D. D. Liposomes: From physics to applications; Elsevier: Amsterdam, 1993. 2. Chonn, A.; Semple, S. C.; Cullis, P. R. J. Biol. Chem. 1992, 267, 18759-18765. 3. Allen, T. Adv. Drug Delivery Rev. 1994, 13, 285-309. 4. Sato, T.; Sunamoto, J. Prog. Lipid Res. 1992, 31, 345-372. 5. Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Biochim. Biophys. Acta 1991, 1066, 29-36.
Acknowledgments We would like to thank Dr. Judith S. Riffle of Virginia Politechnic Institute and State University for useful discussions of polyoxazoline chemistry. Thanks are due to Polymer Chemistry Innovations (State Collage, PA) for providing a sample of Aquazol-5.
JS9504043
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AND
THERESA M. ALLEN‡
Received September 27, 1995, from the †SEQUUS Pharmaceuticals, Inc., 960 Hamilton Court, Menlo Park, CA 94025, and ‡Department of Pharmacology, University of Alberta, Edmonton, AB T6G 2H7, Canada. Final revised manuscript received November 16, 1995. Accepted for publication November 20, 1995X. Abstract 0 Two amphipatic polymers of the poly(2-oxazoline) family, poly(2-methyl-2-oxazoline) (PMOZ) and poly(2-ethyl-2-oxazoline) (PEOZ), were synthesized with the carboxylic group positioned at either the initiation or termination ends of the polymer chains. Distearoylphosphatidylethanolamine was covalently linked to the carboxyl groups of the polymers, resulting in conjugates which incorporate readily into liposomes. Systematic evaluation of plasma clearance kinetics and biodistribution of liposomes containing hydrogenated soy phosphatidylcholine, cholesterol, and 5 mol % the polymer−lipid conjugates in mice revealed the following. Both polymers, PMOZ and PEOZ, exhibited long plasma lifetimes and low hepatosplenic uptake. PMOZ was more effective at decreasing blood clearance rates than PEOZ. The best results, which were quantitatively comparable to the results obtained with the optimized preparations of methoxypolyethylene glycol(PEG)-2000-grafted liposomes, were obtained with formulations containing PMOZ of molecular weight 3260.
Introduction To overcome some of the limitations of liposomes as drug carriers considerable effort has been directed toward extending their plasma lifetimes and diminishing their accumulation in the organs of the mononuclear phagocyte system (MPS, also known as the reticuloendothelial system) (reviewed in ref 1). Since the current understanding of the plasma clearance mechanisms involves interaction of lipid vesicles with plasma proteins,2 modification of liposomal surfaces with the goal to minimize such interactions was one of the main directions of this effort.3,4 Heretofore the best results were achieved by incorporation into liposomal formulations of 3-10 mol % of methoxypolyethylene glycol-2000-distearoylphosphatidylethanolamine (mPEG-DSPE) conjugates.5-8 This results in lipid vesicles with PEG-grafted surfaces. The liposomes of this type exhibit dose-independent pharmacokinetics accompanied with remarkable persistence in vivo.3,9 For example, in humans plasma half-lives of approximately 48 h with enhanced malignant tissue accumulation were observed for PEGylated liposomes.10 The ability of PEG to elicit this effect, when grafted onto the surface of liposomes, has been explained by its chains’ high mobility associated with conformational flexibility and water-binding ability.6,11-13 These properties all contribute to the so-called steric stabilization effect, which results in the well-known propensity of PEG to exclude proteins, other macromolecules, and particulates from its surroundings. It is reasonable to assume that other polymers might possess similar properties and thus, when § Abbreviations: MPS, mononuclear phagocyte system ; PL, phospholipid; POZ, poly(2-oxazoline); PMOZ, poly(2-methyl-2-oxazoline); PEOZ, poly(2-ethyl-2-oxazoline); PEG, polyethylene glycol; mPEG, methoxy-PEG; DSPE, distearoylphosphatidylethanolamine; HSPC, hydrogenated soy phosphatidylcholine; CHOL, cholesterol; DP, dgree of polymerization; TI, tyraminylinulin, GPC, gel permeation chromatography, DMF, N,N′dimethylformamide. X Abstract published in Advance ACS Abstracts, January 1, 1996.
© 1996, American Chemical Society and American Pharmaceutical Association
grafted onto liposomal surfaces, they too should be able to exert similar influences on the vesicles. One class of polymers, poly(2-oxazolines) (POZ), impressed us as particularly promising.14 Similarly to PEG, the backbone of poly(2-oxazoline) is composed of carbon-carbonheteroatom repeating units (see Scheme 1). This suggests similar segmental flexibility of the polymers in solution, which is thought to be of importance for exerting the beneficial “PEG properties” mentioned above.13 Because the pendant N-linked acyl residue is important in influencing the physical properties of POZ, the choice of these groups was limited to the two residues which confer amphipatic properties to these polymers, namely the acetyl and propionyl groups (R ) Me, Et). Indeed, after examination of solubility properties we found a great deal of similarity between PEG and poly(2-methyl-2oxazoline) (PMOZ) and particularly poly(2-ethyl-oxazoline) (PEOZ).15 These similarities in both structure and properties prompted our examination of the two amphipatic polyoxazolines, PMOZ and PEOZ, as modifiers of the liposomal surface. Our preliminary experiments with the POZs showed that both polymers are effective in prolonging liposome circulation time in rats.15 Here we are presenting the results of a systematic evaluation of the plasma clearance kinetics and biodistribution of POZ-grafted liposomes in mice.
Experimental Procedures GeneralsPotassium iodide and methyl p-toluenesulfonate were from Fluka (Ronkonkoma, NY). Ethyl 3-bromopropionate, 2-methyl2-oxazoline, 2-ethyl-2-oxazoline, glutaric anhydride, anhydrous acetonitrile, and chloroform were purchased from Aldrich (St. Louis, MO). Both oxazoline monomers were distilled prior to polymerization over NaOH. A sample of poly(2-ethyl-2-oxazoline) of nominal molecular weight 5000 (Aquazol-5) was obtained from Polymer Chemistry Innovations, Inc. (State Collage, PA). Gel permeation chromatography (GPC) was measured using a Schimadzu-10A instrument equipped with a PL-Gel 5 µm, 1000 Å column (Polymer Laboratories, Inc., Amherst, MA) calibrated with PEG standards in DMF at 40 °C. The data was processed using a GPC program on a Chromatopac C-R7A. 1H-NMR spectra were measured in CDCl or CD OD at 360 MHz on 3 3 a GE instrument by Acorn NMR (Fremont, CA). Hydrogenated soy phosphatidylcholine (HSPC) was obtained from SEQUUS Pharmaceuticals Inc. (Menlo Park, CA). The synthesis of mPEG2000-DSPE was previously described in detail.5,8,16 Cholesterol (CHOL) was purchased from Avanti Polar Lipids (Birmingham, AL). Na125I was purchased from Amersham (Oakville, Canada). Tyraminylinulin was synthesized and 125I-labeled (125I-TI) as previously described.17 Preparation of End-Group-Carboxylated POZssPolymerization reactions were conducted in screw-cap tubes dried at 110 °C, for 24 h prior to their use. The tubes were charged with freshly distilled monomer (5 mL), dry acetonitrile (7 mL) and initiator, purged with nitrogen, sealed, and left stirring in an oil bath at 80 °C for 18 h. Freshly distilled methyl tosylate or alternatively ethyl 3-bromopropionate with 1.1 equiv of KI was used as initiators. Since the 2-oxazoline polymerization process is not self-terminating, also known as living polymerization, the amount of initiator (mole ratio of initiator to monomer) was determined by the desired degree of polymerization (DP). Termination was performed by two equally effective procedures converting the terminal oxazolinium ions into N-(2-hydroxyethyl)
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Scheme 1sSynthesis of polyoxazoline−DSPE conjugates. An asterisk denotes termination by either one of the two methods described in the experimental section. Abbreviations: DCC, dicyclohexylcarbodiimide; HOSu, N-hydroxysuccinimide; Int-X, initiator; X, halide or sulfonate leaving-group residue; TEA, triethyl-amine; -OTs, tosylate. groups. The first method adapted from Kobayashi et al.18 involved addition of solid sodium carbonate and water and heating the reaction mixture at 80 °C for 6 h. Alternatively, a methanolic solution of KOH (0.5 M) was added to the polymerization mixture followed by stirring at 25 °C for 2 h according to the procedure of Riffle and co-workers.19 When ethyl 3-bromopropionate was used as initiator, addition of 2.5 equiv of KOH caused both termination and formation of a carboxyl group by hydrolysis of the ethyl ester at the initiation end of the polymer. Purification of the polymers was accomplished by water dilution of the terminated mixtures to 50 mL and dialysis against 50 mM NaCl, 2% acetic acid, and water (3 × 3 L each) using 3500 molecular weight cutoff Specrta/Por dialysis membrane (Spectrum, Los Angeles, CA). The content of the dialysis bag was lyophilized and then further dried in vacuo over P2O5. Typical recoveries were 70-90%. Polymers initiated with methyl tosylate (including Aquazol-5) were converted into their carboxylated derivatives by reaction with glutaric anhydride. For this purpose POZ was azeotropically dried in benzene (in the case of PEOZ) or acetonitrile (for PMOZ) at a ratio of 10 mL of solvent/g of polymer, concentrated to approximately one-fourth of the initial volume, treated with a 5-fold excess of the anhydride and a 10-fold excess of pyridine, and refluxed for 4 h. The reaction mixtures were evaporated to dryness and dissolved in water and the products recovered by the dialysis, lyophilization, and drying as described above. The polymers were further characterized according to the well-established protocols.18-20 Preparation of POZ-DSPE ConjugatessCarboxylated POZ (0.3 mmol of either glutarate or propionate end groups) was dissolved in chloroform (10 mL) and treated with N-hydroxysuccinimide (0.4 mmol) and dicyclohexylcarbodiimide (0.4 mmol). The solution was stirred overnight at 25 °C and then filtered from precipitated dicyclohexylurea. DSPE (0.25 mmol) was added to the solution followed by triethylamine (2.5 mmol). The reaction mixture was gently warmed up to 40-45 °C for 5-10 min while being stirred. During this time a clear solution was formed as all the DSPE reacted
134 / Journal of Pharmaceutical Sciences Vol. 85, No. 2, February 1996
(confirmed by TLC with ninhydrin staining). The solution was neutralized with acetic acid (2.5 mmol) and evaporated to dryness. The product was purified of unreacted polymer and lower molecular weight reactants using dialysis through 300 000 molecular weight cutoff Spectra/Por CE membrane and recovered as previously described for the PEG-DSPE conjugates.16 The following TLC (silica gel G, visualized with I2 vapor) eluents were used to check the purities of the conjugates: n-propanol/30% aqueous ammonia (7:3), CHCl3/ ethanol/30% aqueous ammonia (3:7:2), CHCl3/ethanol/H2O (80:18:2 and 130:70:8). In the last two solvent mixtures, PMOZ and its conjugates were stationary while PEOZ and its conjugates, as well as free DSPE, were readily separated and detected. The structure of the conjugates was confirmed by 1H-NMR spectra, exhibiting the characteristic peaks of both lipid and polymer components: δ (ppm) 0.9, 1.26, 1.58, 2.28, minor peaks 4-4.5, and 5.2 for DSPE; 2.1 and 3.5 for PMOZ; and 1.13, 2.3-2.4, and 3.5 for PEOZ. The broad polymer peaks often overlapped with minor absorbances, influencing adversely our ability to accurately integrate the spectra. Preperation of LiposomessLiposomes were composed of HSPC: CHOL:mPEG-DSPE (2:1:0.1 molar ratio) or HSPC:CHOL with various amounts of either PEOZ-DSPE or PMOZ-DSPE of various polymer chain lengths. Liposomes were prepared by hydrating dry lipid films in 25 mM HEPES, 140 mM NaCl, pH 7.4 buffer, with the aqueous space label 125I-TI at a lipid concentration of 10 mM. The resulting multivesicular preparations were then passed through 0.10.08 µm polycarbonate membranes (Nuclepore Corp., Pleasanton, CA) using a Lipex extruder (Lipex Biomembranes, Vancouver, B. C.), to give primarily unilamellar vesicles with diameters of 99-112 nm.21,22 The size of the liposomes were determined by dynamic light scattering using a Brookhaven BI90 particle sizer (Brookhaven Instruments, Holtsville, NY). Free 125I-TI was separated from the liposomes by chromatography over a Sepharose CL-4B column with 25 mM HEPES, 140 mM NaCl, pH 7.4 buffer. In Vivo ExperimentssOutbred female CD1(ICR)BR mice were purchased from Charles River Canada (St. Constant, Canada) and maintained in standard housing. Mice (three per group) were given a single bolus iv injection via the tail vein of 0.2 mL of liposomes (0.5 µmol of PL/mouse) containing 2-4 × 105 cpm of 125I-TI. At different times postinjection, animals were anaesthetized with halothane and sacrificed by cervical dislocation. Major organs (liver, spleen, lung, heart, and kidney), blood (100 µL), thyroid, and carcass (remainder of the animal) were collected and counted for 125I label in a Beckman 8000 γ counter. Blood correction factors23 were applied to all samples. The data is expressed as the percentage of counts in each organ relative to the total counts remaining in vivo at each time point.24 Pharmacokinetic parameters of the liposomes were calculated using the polyexponential curve stripping and least squares parameter estimation program RSTRIP (Micromath, Salt Lake City, UT).
Results and Discussion Polyoxazolines are usually synthesized by cationic ringopening polymerization initiated by a variety of alkylating agents and terminated by nucleophiles.14 The nature of the polymerization process allows the use of termination or initiation ends of the polymer chains for placement of functionalized residues for subsequent conjugation with lipids. Using both termination and initiation-based approaches for polyoxazoline functionalization, we prepared several carboxylterminated POZs, as depicted in Scheme 1. Relying on wellestablished literature procedures,18-20 our initial experiments utilized methyl p-toluenesulfonate-initiated polymerization of 2-oxazolines followed by a reaction of hydroxy-terminated polymer with glutaric anhydride for introduction of carboxylic acid group.15 Since both POZs are very hygroscopic and pick up moisture during their recoveries by precipitation, often resulting in pasty solids, we used dialysis followed by lyophilization and drying in vacuo for purification and isolation of polymer derivatives from both polymerization and the following derivatization reactions. Although very simple, this method of POZ isolation is quite time-consuming. In order to minimize the number of manipulations, we developed an alternative approach to preparation of end-carboxylated POZ.
Table 1s Summary of the Polymers and Their DSPE Conjugates Polymer Type
Mn [Mw/Mn]
DPa
Conjugate Type (see Scheme 1)b
MW of DSPE Conjugate
PEOZ
1690 [1.15] 3070 [1.44] 4660 [1.56]c 4420 [1.18] 2020 [1.60] 2290 [1.12] 3260 [1.53] 4330 [1.18] 2000
17 31 47 45 24 27 38 51 45
P P G P G P P P U
2440 3820 5410 5170 2770 3040 4010 5080 2750
PMOZ
mPEG a
Degree of polymerization (DP) was calculated by dividing Mn by the molecular weight of single repeating unit (85 for PMOZ, and 99 for PEOZ). b P ) propionate; G ) glutarate; U ) urethane. c Derivative of Aquazol-5.
Figure 1sThe effect of the type of linkage between the poly(2-oxazoline) polymer and DSPE on the blood elimination of liposomes. (A) Poly(2-ethyl-2-oxazoline) and (B) poly(2-methyl-2-oxazoline) were conjugated to DSPE by either a propionate (P) or glutarate (G) linkage (see Scheme 1). Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (A) (b) PEOZ4420-P−DSPE and (9) PEOZ4660-G−DSPE; (B) (9) PMOZ2020-G−DSPE and (b) PMOZ2290-P−DSPE.
It relied on the use of ethyl 3-bromopropionate as the initiator for 2-oxazoline polymerization. The polymerization reactions proceeded in presence of a 10% molar excess of potassium iodide in dry acetonitrile and resulted in polymers of similar yields and dispersities as in methyl tosylate-initiated reactions. Upon termination of the polymerization process with either aqueous carbonate or methanolic potassium hydroxide, the ethyl propionate end group was converted into a free carboxyl residue. The molecular weights of the synthesized polymers were in the range Mn 1500-5000 Da with low dispersity (Mw/Mn 1.1-1.6), as determined by GPC.25 The end-carboxylated polyoxazolines, prepared by both functionalization approaches shown in Scheme 1, were converted in situ into their succinimidyl esters and then coupled to amino group of DSPE in chloroform under conditions previously used for preparation of PEG-lipids.26 We characterized the products by TLC and 1H-NMR spectra. Some properties of the polyoxazolines and their conjugates are summarized in Table 1. Liposomes containing the POZ conjugates listed in Table 1 were prepared (105 ( 6 nm) with the standard composition HSPC/CHOL/POZ-DSPE 2:1:0.1. Thus the polymer-lipid conjugate was always present at 5 mol % of phospholipid content. The POZ-grafted liposomal preparations were labeled with 125I-tyraminylinulin and then injected into mice for determination of their longevity in circulation and their biodistribution parameters, according to previously described methodologies.5,27 As can be seen from Figure 1, polymers of the same (or similar) molecular weights grafted onto liposomes through either P- or G-type of linkages positioned at either initiation or termination ends, respectively, produce approximately the same plasma clearance rates. Although aliphatic ester-containing conjugates similar to G-type POZDSPE are known to be labile in aqueous buffers,28 the
hydrolysis rates are usually too slow to have an effect on the clearance rates.29 Since the results obtained with either Por G-type of lipid conjugates were essentially the same and since the P-type of conjugates was more convenient to prepare, we continued our experimentation with P-type of conjugates only. Figure 2 illustrates the relationship between circulation times and molecular weights of the grafted polymers. Among the PEOZ-containing formulations, variation of molecular weight of the grafted polymers produced hardly any changes in the longevity of the liposomes in blood circulation (Figure 2A). Preparations containing PMOZ-3260 showed the best results among PMOZ liposomes (Figure 2B), with PMOZ-4330grafted vesicles producing only slightly inferior results. Comparison of the best-performing PMOZ liposomes with preparations containing mPEG-2000, previously found to be optimal among PEG-grafted liposomes,5,8 and with PEOZ4420-containing liposomes is shown in Figure 3. Although, as shown in Figure 2A, there was no significant difference between the tested PEOZ preparations, PEOZ-4420 was chosen as “the best” based on slightly higher blood levels of its liposomes at later time points (24 and 48 h). The comparison of the three preparations (Figure 3) demonstrates that lipid vesicles grafted with PMOZ-3260 produced a similar pharmacokinetic curve to liposomes containing mPEG-2000. While the results obtained with PEOZ-4420-modified vesicles were not quite as favorable, this preparation still can be viewed as long-circulating in comparison to “classical” (nonPEGylated) liposome formulations. The biodistribution results summarized in Table 2 showed good correlation between longevity in circulation and low uptake by liver and spleen. The biodistribution results also confirm the similarity between the two longest circulating formulations, containing mPEG2000 or PMOZ-3260. The pharmacokinetic parameters of the polymer-grafted liposomes and their “classical” counterparts (i.e., not containing polymer) are given in Table 3. “Classical” liposomes were either neutral or contained 5 mol % of phosphatidylglycerol (PG) to provide the same amount of negative charge carried by the polymer-grafted liposomes. The mean residence time, half-life, and the area under concentration-time curve (AUC) are significantly higher for the polymercontaining liposomes compared to liposomes lacking polymer. The elimination rate constant is significantly lower for the polymer-containing liposomes. Note that the degree of polymerization (DP) values of the best performing polymers, mPEG-2000, PMOZ-3260, and PMOZ-4330, are in 38-51 range (Table 1). This might be important since DP determines the number of “bending units” and as such is directly related to the degree of conformational freedom of polymer chains. The latter parameter is thought to be consequential to the beneficial influence of the polymers on liposome blood clearance.6,11-13,15 The observation that the higher molecular weight PMOZ conjugate was cleared from
Journal of Pharmaceutical Sciences / 135 Vol. 85, No. 2, February 1996
Table 2sTissue Distributions of Polymer-Grafted Liposomes in Micea Polymer
Blood
Liver
mPEG-2000 PMOZ-3260 PEOZ-4420
77.3 ± 1.1 82.2 ± 3.0 61.3 ± 6.7
11.8 ± 0.9 10.7 ± 1.9 21.1 ± 3.0
mPEG-2000 PMOZ-3260 PEOZ-4420
27.2 ± 8.3 22.1 ± 4.0 9.7 ± 3.0
22.2 ± 1.0 32.1 ± 1.0 44.7 ± 5.8
Spleen 2 h postdose 1.7 ± 0.6 1.3 ± 0.4 5.3 ± 1.0 24 h postdose 4.8 ± 0.5 3.8 ± 0.7 9.2 ± 2.2
Lung
Heart
Kidney
Carcass
0.6 ± 0.4 0.3 ± 0.3 0.3 ± 0.1
0.5 ± 0.1 0.8 ± 0.1 0.4 ± 0.1
1.7 ± 0.8 1.9 ± 0.3 1.5 ± 0.5
6.5 ± 0.5 2.8 ± 3.7 10.0 ± 5.2
0.2 ± 0.0 0.5 ± 0.1 0.3 ± 0.1
0.6 ± 0.2 0.4 ± 0.1 0.3 ± 0.1
3.2 ± 0.4 3.5 ± 0.7 2.2 ± 0.4
41.5 ± 8.8 36.9 ± 2.0 32.5 ± 4.9
a Liposomes (ca. 100 nm) were composed of HSPC:CHOL (2:1, molar ratio) containing 5 mol of either mPEG-2000−DSPE (n ) 3), PMOZ-3260−DSPE (n ) 3), or PEOZ-4420−DSPE (n ) 9). Both polyoxazoline compounds contained the proprionate linkage. Tissue distributions were determined for 125I-tyraminylinulin-labeled liposomes (0.5 µmol of phospholipid per mouse) at 2 and 24 hours after iv injection. Mean ± SD.
Figure 3sComparison between polyethylene glycol and the polyoxazolines on elimination of liposomes from blood. Liposomes (ca. 100 nm) were composed of HSPC:CHOL, 2:1 molar ratio with 5 mol % polymer−lipid. Both POZ were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (9) mPEG2000−DSPE, (b) PMOZ3260-P−DSPE, and (2) PEOZ4420-P−DSPE. Table 3sPharmacokinetic Parametersa
Figure 2sThe effect of polymer molecular weight of (A) poly(2-ethyl-2-oxazoline)− DSPE and (B) poly(2-methyl-2-oxazoline)−DSPE on blood elimination of liposomes. Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Polymer lipids were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per the methods. Key: (A) (2) PEOZ4420P−DSPE, (1) PEOZ3070-P−DSPE, and (9) PEOZ1690-P−DSPE; (B) ([) PMOZ4330P−DSPE, (b) PMOZ3260-P−DSPE, and (9) PMOZ2290-P−DSPE.
blood circulation slightly faster than the PMOZ-3260 analog might be explained by the known propensity of lipid-linked polymer chains of increased length to dissociate faster from lipid bilayers than their shorter chain counterparts.30 A similar rationale was used to explain the differences between mPEG-2000- and mPEG-5000-grafted liposomes.29 Since, the rate of dissociation and thus bilayer retention of polymerlipids has also been shown to be hydrophobic-anchor-dependent, we chose to prepare the POZ conjugates from DSPE, previously shown to be the optimal lipid anchor.31 Finally, we looked at the relationship between the plasma persistence of formulations and concentration of POZ-lipid. As can be seen from Figure 4, at 24 h postinjection variation of the mol percentage of either polymer conjugate had a noticeable effect. For PEOZ-4420-grafted liposomes, increasing the polymer content from 2 to 5 mol % resulted in a more than doubling of the blood levels, with no further increase at higher mol percentages of the conjugates. PMOZ-containing liposomes also exhibited consistently higher blood levels with 136 / Journal of Pharmaceutical Sciences Vol. 85, No. 2, February 1996
Liposome Composition
MRTb (h)
AUC0-∞c (nmol h/mL)
k10d (h-1)
t1/2R (h)
t1/2β (h)
HSPC:CHOL:mPEG2000−DSPE HSPC:CHOL:PMOZ3260-P−DSPE HSPC:CHOL:PEOZ4420-P−DSPE HSPC:CHOL HSPC:CHOL:PG
23.5 21.7 13.8 9.0 5.3
4761 4388 2267 2031 1271
0.06 0.06 0.10 0.15 0.24
3.16 5.1 0.77 0.01 0.05
17.7 17.8 9.9 6.2 3.7
a Mice were given a single bolus iv injection, via the tail vein, with liposomes (0.5 µmol of phospholipid, ca. 100 nm in diameter). Liposomes were composed of HSPC:CHOL at a 2:1 molar ratio with PG, POZ−DSPE or mPEG−DSPE present at 5 mol % of phospholipid. Blood samples were taken at various times up to 48 h postinjection. All pharmacokinetic parameters were calculated from a twocompartment open model (biexponential curve fit) using the program RSTRIP (Micromath, Salt Lake City, UT). b Mean residence time. c Area under the blood concentration vs time curve. d Elimination rate constant from the central compartment.
increasing content of polymer-lipid in the formulations. At all polymer concentrations, PMOZ-containing liposomes yielded higher blood levels than their more hydrophobic PEOZ counterparts. Thus the difference between the effects of PMOZ or PEOZ on liposome circulation times became more pronounced with increasing mol percentage of polymer-lipid components in the formulations, until the effect reached saturation between 5-10 mol %. It is pertinent to note that during early attempts to prepare long-circulating liposomes it was observed that GM1-containing formulations32 exhibited circulation longevity in mice but not in rats.33 Although the mechanism of liposomal surface protection by grafted synthetic polymers, like POZs, must be different from that of GM1, it was important to confirm that polyoxazoline-grafted liposomes exhibited plasma longevity
Figure 4sThe effect of increasing the poly(2-oxazoline) content in liposomes on blood elimination at 24 hours postinjection. Liposomes (ca. 100 nm) were composed of HSPC:CHOL:POZ−DSPE, 2:1:0.1 molar ratio. Polymer lipids were conjugated to DSPE by a propionate linkage. Mice were given a single bolus iv injection of 500 nmol of liposomes, via the tail vein. Biodistributions were performed as per methods. Key: (b) PMOZ3260-P−DSPE and (2) PEOZ4420-P−DSPE.
and low hepatosplenic uptake in more than one species. From the experimental observations reported here and in our earlier communication,15 it appears that POZs convey long circulation properties to liposomes in both rats and mice, similar to PEG. Quantitatively the effect is also comparable to that of PEG. Although there have been other attempts to utilize synthetic polymers other than PEG for preparation of long-circulating liposomes,34-36 to the best of our knowledge to date only PMOZ liposomes in mice and both PMOZ and PEOZ liposomes in rats exhibited the same ability to avoid MPS uptake and to extend blood lifetimes as their mPEG-containing counterparts. Although there is a considerable amount of literature on POZs14 thus far only a few papers dealt with applications of these polymers to biomedical problems.20,37-40 These include the evaluation of PMOZ as a promising drug carrier.37 Our observations that at 48 h postdose the mice showed no signs of toxicity, abnormal behavior, or organ abnormalities are in agreement with the previously published observations suggesting the low toxicity and good biocompatibility of POZs.
Conclusions Our results demonstrate that two amphipatic polymers of the poly(2-oxazoline) family, PEOZ and PMOZ, are efficient in conveying long circulation properties to liposomes in mice. This finding, combined with the previously reported ability of these polymers to cause MPS evasion and longevity in circulation in rats, suggests that, similar to PEG, the effect maybe species-independent. Judging by the plasma clearance kinetics and biodistribution in mice, PMOZ-3260-grafted liposomes perform as well as mPEG-2000 liposomes, while PEOZ-containing preparations are somewhat inferior. To our knowledge, poly(2-methyl-2-oxazolines) linked to DSPE are the only polymers conjugates able to convey long circulation and low hepatosplenic uptake to liposomes to the same extent as PEG. In light of these encouraging initial results, it is prudent to verify whether the usefulness of the amphipatic POZs also extends to other in vivo applications: conjugates of proteins, biocompatible surfaces, etc.
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References and Notes 1. Lasic, D. D. Liposomes: From physics to applications; Elsevier: Amsterdam, 1993. 2. Chonn, A.; Semple, S. C.; Cullis, P. R. J. Biol. Chem. 1992, 267, 18759-18765. 3. Allen, T. Adv. Drug Delivery Rev. 1994, 13, 285-309. 4. Sato, T.; Sunamoto, J. Prog. Lipid Res. 1992, 31, 345-372. 5. Allen, T. M.; Hansen, C.; Martin, F.; Redemann, C.; Yau-Young, A. Biochim. Biophys. Acta 1991, 1066, 29-36.
Acknowledgments We would like to thank Dr. Judith S. Riffle of Virginia Politechnic Institute and State University for useful discussions of polyoxazoline chemistry. Thanks are due to Polymer Chemistry Innovations (State Collage, PA) for providing a sample of Aquazol-5.
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