Association of Hydrophobically-Modified PEG with Liposomes

Chapter 8

Polymer-Protected Liposomes: Association of Hydrophobically-Modified PEG with Liposomes 1

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Debra T. Auguste , Robert K. Prud'homme *, Patrick L. Ahl , Paul Meers , and Joachim Kohn 2,5

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Downloaded by CORNELL UNIV on July 7, 2012 | http://pubs.acs.org Publication Date: February 16, 2006 | doi: 10.1021/bk-2006-0923.ch008

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Department of Chemical Engineering, Princeton University, Princeton, NJ 08544 Elan Pharmaceuticals, One Research Way, Princeton, NJ 08540 Department of Chemistry, Rutgers, the State University of New Jersey, Piscataway, NJ 08854 Current address: BioDelivery Systems International, 4 Bruce Street, Newark, NJ 07103 Current address: Transave, 11 Deer Park Drive, Suite 117, Monmouth Junction, NJ 08852 *Corresponding author: [email protected] 2

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We demonstrate the impact of cooperative interactions on liposome protection by incorporating multiple hydrophobic sites on poly(ethylene glycol) (PEG) polymers. Our hydro phobically-modified PEGs (HMPEGs) are comb-graft copoly mers with precisely alternating, monodisperse PEG blocks (MW= 6, 12, or 35 kDa), bonded to C18 stearylamide hydro phobes. Cooperativity is controlled by varying the extent of oligomerization at a constant ratio of PEG to stearlyamide. The association of polymer with liposomes increases with the degree of oligomerization; equilibrium constants for 6 kDa PEG increases from 6.1±0.8 (mg/m )/(mg/ml) for 3 loops to 78.1±12.2 (mg/m )/(mg/ml) for 13 loops. In addition, HMPEG6k-DP3 (with three 6 kDa loops) results in superior, irreversible protection from complement protein binding than DSPE-PEG5k after 12 hours. 2

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© 2006 American Chemical Society

In Polymeric Drug Delivery I; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction Liposomes, or vesicles, are concentric lipid bilayers separated by aqueous volumes [1]. Because they are biodegradable, nontoxic, and able to noncovalently encapsulate molecules (i.e., chemotherapeutic agents, hemoglobin, imaging agents, drugs, and genetic material) within their 100-200 nm diameter interior, liposomes have advantages over other drug delivery methods [2]. In addition, liposomes can target specific sites for drug delivery [3]. Clearance by macrophages, as a result of immune recognition, can localize liposomes in sites of inflammation. Enhanced permeability of immature tumor vasculature to particles with sizes less than 100-780nm provides localization in tumors [4]. Controlled release of agents from liposomes can be exploited to maintain therapeutic drug concentrations in the bloodstream or in local areas. Other benefits of encapsulation are protection of the drug from degradation and efficient intracellular delivery [5]. Liposomes are typically categorized in one of four categories: (1) conventional liposomes, which are composed of neutral and/or negativelycharged lipids and often cholesterol; (2) long-circulating liposomes, which incorporate PEG covalently bound to a lipid; (3) immunoliposomes, used for targeting where antibodies or antibody fragments are bound to the surface, and (4) cationic liposomes, that are used to condense and deliver DNA [5]. Liposomes are characterized by surface charge, size, composition, and number and fluidity of the lamellae [6]. Certain lipids have been shown to affect the mechanics of liposomes by making them more rigid (i.e., cholesterol) or more fiisogenic (i.e., phosphatidylethanolamine and N-dodecanoyl-phosphatidylethanolamine) [7,8]. These parameters have been investigated to increase liposome accumulation in sites of interest, which is typically hindered by immune recognition [3,8-16]. Immune recognition results in liposome accumu lation in the liver, kidney, and spleen, which are the organs whose primary responsibility is to remove toxins and foreign agents from the bloodstream [12]. Some scientists have focused on liposomal delivery to these organs based on formulations that cause immune recognition [11,17]. For delivery to tumors, evasion of the immune system is vital. Increased circulation has been shown to correspond to an increase in liposome accumulation in tumors [12,18]. Looking at nature, scientists studied natural cell membranes to devise methods to reduce immune recognition. Glycolipids, receptors, and eventually hydrophilic polymers were covalently attached to lipids and incorporated into liposomes [2,3,19-21]. The most advantageous liposome formulation (the "Stealth" liposome) integrated a PEG covalently bound to a lipid (i.e., DSPEPEG5k) in the liposome formulation [2]. A range of PEG molecular weights



97 were investigated. Ultimately, 2-5 kDa showed the strongest ability to reduce immune recognition [22]. Blume and Cevc demonstrated that incorporation of at least 2.5 mol% DSPE-PEG5k increased circulation times from 0.47 to 8 hours [23]. It is postulated that the limitation of DSPE-PEG5k for extending the circulation times arises from deprotection of the liposomes by partitioning of the PEG-lipid off of the surface. The PEG layer produces a steric barrier to protein binding. Senior showed a decrease in the rate of protein adsorption onto liposomes incorporating DSPE-PEG5k [16]. In parallel, Chonn et al showed greater protein binding on bare liposomes, which correlated with shorter circulation times [24]. Evidence that immune recognition was a result of complement protein adsorption was demonstrated by Liu et al [14]. Complement proteins are known to cause a cascade of events that result in immune recognition. Therefore, we employ an automated in vitro assay that measures the ability of the HMPEG polymers to shield liposomes from complement interactions. We present multiply attached polymers as a means for constructing polymer protected liposomes. The method exploits current interest in cooperativity, where multiple, relatively weak binding leads to strong overall association. The concept is established by a series of PEG-based comb copolymers with concatenated PEG chains having hydrophobic anchoring groups between the linked PEG chains. These polymers allow the direct comparison of binding in relation to protection with polymers with the same ratio of PEG to hydrophobe but with varied oligomerization and polymers with the same degree of oligomerization but with different ratio of PEG to hydrophobe. The HMPEG polymers provide substantial benefits over PEGylated lipids. The ability to add the soluble polymer to preformed liposomes permits greater flexibility in manufacturing and tailoring of liposome formulations. Also, PEGylated lipids are limited in the molecular weight of PEG and in the amount able to be incorporated into the liposome. The former occurs due to partitioning and the latter results from phase transitions. With multiple binding interactions, higher molecular weight PEGs can be bound at higher binding affinities. The HMPEG polymers we investigate in this study are comb-graft copolymers with precisely alternating, monodisperse PEG blocks (Mw= 6, 12, or 35 kDa), bound to C18 stearylamide hydrophobes with 3 to 13 hydrophobic anchors per chain. We report the ability of these polymers to associate with fiisogenic liposomes and shield from complement binding. We benchmark our results against DSPEPEG5k, which has been studied previously.


98

Materials and Methods


Liposome Preparation N-C12-DOPE:DOPC (7:3, mokmol) liposomes were prepared as described by Shangguan et al.[7]. Lipids solubilized in chloroform at 25 mg/ml were added to a vial at a final molar mass of 80 umol. Chloroform was removed under reduced pressure using a Buchi RE 111 Rotovapor and 461 water bath (Biichi Labortechnik AG, Flawil, Switzerland), then left under vacuum overnight to remove residual solvent. The lipid bilayer film was hydrated with a TES buffer solution (10 mM TES, 150 mM NaCl, 0.1 mM EDTA, pH 7.4), vortexed, subjected to five cycles of freezing in liquid nitrogen and thawing in a room temperature water bath. The lipid solution was extruded ten times through a 0.2 um polycarbonate membrane filter at 250 psi using a 10 ml Lipex extruder (Northern Lipids, Inc. Vancouver, BC, Canada). The liposomes were stored at 4°C. To aid in separation from free polymer in solution, the liposome density was increased by encapsulating a sucrose solution (10 mM TES, 250 mM sucrose, pH 7.4) and a fluorescent marker (DiD) was added for visibility. Unencapsulated sucrose was removed by dialysis at 4°C overnight against the TES buffer using a Slide-A-Lyzer cassette with a 10k MWCO. Sucroseencapsulating liposomes containing DSPE-PEG5k were prepared similarly however the amount of DSPE-PEG5k (x) added was subtracted from the mol ratio of DOPC (2.9-x), keeping the ratio of DiD and N-C12-DOPE constant. Therefore, the composition of liposomes incorporating DSPE-PEG5k is denoted as N-C12-DOPE:DOPC:DSPE-PEG5k:DiD (7:(2.9-x):x:0.1, mol:mol:mol:mol). Liposome concentration was determined by the phosphate assay as described by Chen et a/.[25]. The liposome radii and polydispersity were determined by quasi-elastic light scattering using a NICOMP 270 submicron particle sizer (NICOMP Instruments, Goleta, CA). The number-averaged diameters for sucrose-encapsulating and buffer-encapsulating liposomes were 62.1 ± 16.4 nm and 112.8 ± 28.5 nm, respectively. Adsorption HMPEG polymers (for polymer description see Table I) were equilibrated with liposomes at 1.4mM lipid in 0.5 ml TES buffer. Polymer was then added, either solubilized in TES buffer or as dry mass, to achieve the desired concentration. For liposomes with DSPE-PEG5k, four liposome formulations were prepared with increasing amounts of polymer coverage (0.14, 0.28, 0.70, and 2.4 times T*, i.e., full surface coverage as calculated from the dimensions of the PEG loops [26]. The DSPE-PEG5k containing liposomes were diluted to 1.4mM


99 lipid in TES buffer as described above. Each sample was vortexed and allowed to equilibrate overnight at room temperature in an Eppendorf 5436 Thermomixer (Brinkman Instruments, Westbury, NY, USA). After 24 hours, an initial fraction of 200 ul was removed. The remaining 0.3 ml was centrifuged at 21000xg in the Eppendorf 5417r Centrifuge (Brinkmann Instruments, Westbury, NY, USA) at 4°C. The supernatant was extracted. Phosphate and PEG assays were completed on the supernatant and initial fractions to detect the amount of PEG per lipid.


Table I. Description of hydrophobically-modified PEG (HMPEG) polymers. Polymer

0.88

r\ * nm ) anchor mol% 0.89

HMPEG6k-DP3

4

50

42

Polymer Area, xl0 m 7.9

HMPEG6k-DP13

24

43

112

35.0

0.53

1.21

HMPEG12k-DP2.5

3

75

48

13.3

0.60

0.40

HMPEG 12k-DP5

8

65

106

26.5

0.66

0.53

HMPEG35k-DP2.5 DSPE-PEG5k

3 1

128

138

38.8

0.59

0.14

54

5781

2.3

0.42

1.55

N

b

A

MW, kDa

1 7

2

r*, mg/m

1

2

2

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To convert T* (mg/m ) to r *, determine the moles polymer per 4.7xl0" moles of lipid then multiply by the number of hydrophobes (DP-1) and 100 to obtain the mol% hydrophobes per lipid, or Ch *. hm

m

The association constant K (see Table II), was determined by the initial slope (first 4 data points) of each adsorption profile, such that 2

K

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rfT _ (mg/m ) d[C (Jree)\ (mg/ml)

)

(1

p

PEG Assay The concentration of PEG was quantified by an assay described by Baleux [27], wherein 25 ul of an iodine-potassium iodide solution (0.04 M I , 0.12 M KI) was added to 1 ml of a diluted sample. Samples were adjusted to an optimal adsorption range (0.1

Association of Hydrophobically-Modified PEG with Liposomes

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