Micelles as Intermediates in the Preparation of ProteinâLiposome

Bioconjugate Chem. 2006, 17, 438−450

438

Micelles as Intermediates in the Preparation of Protein-Liposome Conjugates Jennifer Takasaki and Steven M. Ansell* Inex Pharmaceuticals Corporation, 100-8900 Glenlyon Parkway, Burnaby, BC, V5J 5J8, Canada. Received February 24, 2005; Revised Manuscript Received November 21, 2005

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of proteinliposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible proteinliposome-based drug formulations.

INTRODUCTION The attachment of proteins to the surface of liposomes in order to alter their in vivo behavior is an area of continuing interest. These proteins might serve a wide range of functions, with the most common application being use as targeting vectors (1) for liposomal drug delivery systems. More currently, interest is increasing in the use of liposome surface-bound proteins and peptides as antigens in immunological applications (2-4). Traditionally proteins have been conjugated to liposomes using maleimide-sulfhydryl coupling chemistry (5). Typically the protein is thiolated using a heterobifunctional cross-linker and subsequently coupled to the surface of liposomes containing a maleimide lipid as one of the lipid components. In most reports the reagent SPDP1 has been used as the cross-linker. More recently a few reports using 2-IT have appeared (6, 7). Early work in the field involved coupling proteins directly onto the surface of the liposome. Many problems were experienced with this approach due to cross-linking reactions between protein-liposome conjugates and additional liposomes, leading to aggregation (8). Since size is an important parameter in determining the clearance behavior in vivo (9), these liposome-protein conjugates often suffered from poor pharmacokinetics in vivo. Many years ago we resolved this problem * To whom correspondence should be addressed. 201-2010 Weighth Ave, Vancouver, B. C., V6J 1W5, Canada. Phone (604) 733-1231. E-mail: [email protected]. 1 Abbreviations: 2-IT, 2-iminithiolane; SPDP, N-succinimidyl 3(2-pyridyldithio)propionate; PEG, poly(ethylene glycol); POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; chol, cholesterol; DSPE-MPB, N-4′-(4′′-maleimidophenyl)butanoyl-1,2-distearoyl-snglycero-3-phosphoethanolamine; DSPE-ATTA2-MPA, N-(N′-(N′′-(3′′′maleimidopropionoyl)-14′′-amino-3′′,6′′,9′′,12′′-tetraoxatetradecanoyl)14′-amino-3′,6′,9′,12′-tetraoxatetradecanoyl)-1,2-distearoyl-sn-glycero3-phosphoethanolamine; OGP, octyl β-D-glucopyranoside; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; HBS, HEPES-buffered saline; DTT, 1,4-dithio-DL-threitol; ATTA, 14-amino3,6,9,12-tetraoxatetradecanoic acid; DSPE, 1,2-distearoyl-sn-glycero3-phosphoethanolamine; DCC, N,N′-dicyclohexylcarbodiimide; NHS, N-hydroxysuccinimide; TFA, triflouroacetic acid; MePEGS-2000DSPE, N-(4′-O-(ω-monomethoxypoly(ethylene glycol)2000)succinoyl)1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

by incorporating small amounts of PEG-lipids in the liposome formulations to act as steric barriers to inhibit the cross-linking reaction (10). However, including these “steric barrier” molecules in the bilayer also reduced the overall conjugation efficiency of the protein to the bilayer surface. This problem was partially resolved by using maleimide lipids (or other reactive lipids) in which the reactive function and the lipid anchor were separated by a PEG chain (11). The steric inhibition of the conjugation step seen in our early work was partially abolished when the reactive function was placed outside of the steric barrier created by the PEG-lipids in the formulation. Recently work done in Allen’s laboratory has demonstrated that maleimide- or hydrazide-PEG-lipids in pure or mixed micelles could be reacted with thiolated or oxidized proteins, respectively. The products from those reactions could then be exchanged into bilayers when they were mixed with conventional liposomes (12). We believe that the critical aspect of these recent publications is not that the protein is conjugated to the terminus of a PEGlipid, but rather the fact that the process is based on a micelle intermediate. By performing the actual conjugation reaction remote from the liposome surface we can potentially obtain a system that has a high degree of reproducibility. In fact it may not be necessary to have the PEG tether present at all, if the reaction is done under the appropriate conditions. With this hypothesis in mind we set out to explore the range of utility of micelle-based conjugation methods and to characterize the parameters that might affect the outcome of those reactions. We determined that micelle-based reactions can be performed on a wide range of micelle compositions, and in fact that large steric barrier molecules are unnecessary and frequently undesirable components in the formulation. The conditions that lead to aggregation were characterized, allowing us to develop formulations with minimal aggregation without resorting to the use of additional steric barrier molecules. Application of this micelle-based technology led to the development of reproducible, simple, and scaleable methods for preparing liposomeprotein conjugates. There are two general methods which can be used for preparing liposomes with proteins attached to their surfaces. Ultimately the protein is conjugated to a lipid anchor, and this

10.1021/bc050051r CCC: $33.50 © 2006 American Chemical Society Published on Web 01/12/2006

Micelle-Mediated Conjugation

Bioconjugate Chem., Vol. 17, No. 2, 2006 439

Figure 1. Examples of protocols involving active and passive coupling techniques. In this work active coupling refers to methods in which the lipid-protein conjugation reaction occurs at the liposome surface, and passive coupling refers to methods in which the lipid-protein conjugation reaction occurs remote from the liposome surface.

can be done either at the surface of a preformed liposome with the appropriate active lipid already present, or it can be done remotely and then introduced to the liposome at a later point, using a variety of methods. We are using the terms “active” and “passive” coupling in this work when referring to the two types of conjugate formation. Examples of these two types of coupling procedures are presented in Figure 1. “Active coupling” applies to procedures where the chemical linkage occurs at the liposome surface. There are a number of ways to produce a liposome with an active lipid incorporated in the membrane structure. For example, liposomes could be prepared directly where the active lipid is one of the component lipids (8). Alternatively, liposomes could be prepared without the active lipid present. The active lipid may then be exchanged into the outer leaflet of the bilayer from micelles. Both methods would result in liposomes with reactive functions on their surfaces, available for conjugation with a suitably derivatized protein. The former method is the one generally used in the literature for the preparation of protein-liposome conjugates. “Passive coupling” applies to procedures where the protein is conjugated to the lipid anchor away from the liposome surface. Generally in such reactions a micelle serves as the platform for performing the conjugation reaction since lipids and proteins are usually not soluble in the same solvents. Once formed, the protein-lipid conjugate could then be incorporated into the bilayer during liposome formation, either by cohydration of dried lipid/conjugate, or by detergent dialysis. Alternatively, the micelles on which the conjugates were formed could be exchanged into preformed liposomes (12). We have used the latter method as our starting point for this work.

EXPERIMENTAL PROCEDURES Materials. DSPE was obtained from Genzyme Pharmaceuticals, Cambridge, MA. DSPE-MPB was obtained from Northern lipids Inc, Vancouver, Canada. 2-IT was obtained from SigmaAldrich. Synthesis of 11-Azido-3,6,9-trioxaundecanol 1. A solution of tetraethylene glycol (1.2 kg) and methanesulfonyl chloride (400 mL) in benzene (2 L) was cooled in an ice bath. Triethylamine (716 mL) was slowly added with stirring. The reaction mixture was stirred at room temperature for 2 h and filtered and the solvent removed on a rotovap. The oil was diluted with water (100 mL). Saturated sodium bicarbonate solution was then added until the solution tested neutral using pH paper. The mixture was diluted with ethanol (1.6 L). Sodium azide (270 g) was then slowly added as a slurry in water (500 mL). The reaction mixture was heated to 60 °C with stirring for 2 h. Water (5 L) was added to the reaction mixture, and the resultant solution was washed with diethyl ether. The aqueous fraction was then extracted with dichloromethane (3 passes, 500 mL). The combined organic fraction was washed with water (250 mL), which in turn was extracted with methylene chloride (250 mL). The combined methylene chloride fractions were dried over anhydrous magnesium sulfate and filtered, and the solvent was removed on a rotovap, yielding ∼400 g of 1. Synthesis of 14-Azido-3,6,9,12-tetraoxatetradecanoic Acid 2. Finely powdered potassium bromoacetate was prepared by neutralizing a solution of bromoacetic acid in water with potassium hydroxide and lyophilizing the resultant solution. A portion of this product (270 g) was added to a solution of 1 (252 g) in toluene (750 mL) in a water-jacketed reactor. The suspension was vigorously stirred with a mechanical stirrer at 45 °C, and four portions of sodium hydroxide pellets (4 × 40

440 Bioconjugate Chem., Vol. 17, No. 2, 2006

g) were added at 15 min intervals. The reaction was allowed to proceed for an hour and then quenched by the addition of water (500 mL). The two phases were allowed to separate and the aqueous phase run off. The toluene phase was washed with 5% sodium hydroxide. The two aqueous phases were combined and washed with methylene chloride. The resultant aqueous phase was acidified to ∼pH 3-4, and extracted with methylene chloride. The organic fraction was dried over magnesium sulfate and filtered and the solvent removed on a rotovap, yielding crude 2 as a pale yellow oil (∼120 g). Synthesis of Ethyl N-(N′-BOC-14′-amino-3′,6′,9′,12′-tetraoxatetradecanoyl))-14-amino-3,6,9,12-tetraoxatetradecanoate 4. Ethyl N-(14′-amino-3′,6′,8′,12′-tetraoxatetradecanoyl)14-amino-3,6,9,12-tetraoxatetradecanoate 3 was prepared as previously described (13). A solution of 3 (30 g) in methylene chloride (100 mL) was treated with di-tert-butyl dicarbonate (13.2 g) and triethylamine (8.5 mL) at room temperature for 1 h. The solution was washed with dilute hydrochloric acid, dried over anhydrous magnesium sulfate,and filtered and the solvent removed on a rotovap. Yields ∼30 g of 4 as a colorless oil. Synthesis of N-(N′-(N′′-(tert-Butoxycarbonyl)-14′′-amino3′′,6′′,9′′,12′′-tetraoxatetradecanoyl)-14′-amino-3′,6′,9′,12′tetraoxatetradecanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine 5. A solution of 4 (5 g) in aqueous sodium carbonate (4.5 g in 100 mL) was warmed at 60 °C for 30 min with stirring. The solution was washed with methylene chloride, acidified to ∼pH 3 with hydrochlorics acid, and extracted with methylene chloride. The organic phase was dried over magnesium sulfate and filtered and the solvent removed. The residue was dissolved in alcohol free chloroform (50 mL). N-Hydroxysuccinimide (1.65 g) was added, followed by a solution of dicyclohexylcarbodiimide (2.90 g) in alcohol free chloroform (50 mL). The solution was stirred at room temperature for 20 min and then filtered. 1,2-Distearoyl-sn-glycero-phosphethanolamine (DSPE, 4.5 g) was added, and the solution was heated in a water bath at 60 °C until the DSPE had dissolved. Triethylamine (3 mL) was added and the solution stirred at room temperature for 1 h. The solution was filtered, diluted with water, and extracted with methylene chloride. The organic phase was washed with dilute hydrochloric acid and dried over anhydrous magnesium sulfate and the solvent removed on a rotovap. The product was purified using a preparative HPLC, yielding 5 as a colorless solid (7.6 g). Synthesis of N-(N′-(N′′-(3′′′-Maleimidopropionoyl)-14′′amino-3′′,6′′,9′′,12′′-tetraoxatetradecanoyl)-14′-amino3′,6′,9′,12′-tetraoxatetradecanoyl)-1,2-distearoyl-sn-glycero3-phosphoethanolamine. 6. Trifluoroacetic acid (4 mL) was added to solid 5 (2.75 g) and stirred at room temperature for 30 min. The excess trifluoroacetic acid was removed on a rotovap and the residue dissolved in distilled water (50 mL), neutralized with aqueous sodium hydroxide, and extracted with methylene chloride. The organic fractions were dried over anhydrous magnesium sulfate and filtered and the solvent removed. 3-Maleimidopropanoic acid (0.41 g) was dissolved in chloroform (20 mL) and treated with NHS (0.42 g) and DCC (0.71 g). The solution was stirred at room temperature for 20 min and filtered. The filtrate was added to a solution of the previously obtained crude product in chloroform (30 mL). Triethylamine (2 mL) was added and the solution stirred at room temperature for 1 h. The solution was filtered, diluted with water, and extracted with methylene chloride. The organic fractions were washed with dilute hydrochloric acid, dried over anhydrous magnesium sulfate, and filtered, and the solvent was removed. The residue was purified by HPLC, taken up in benzene, and lyophilized, yielding 6 (1.7 g) as an off white powder. The product appeared as a single spot on TLC analysis and showed

Takasaki and Ansell

no evidence of primary amines when stained with a flourescamine/acetone solution and viewed under UV light. Preparation of Liposomes. Lipid mixtures composed of POPC/chol (55:45) were dissolved in chloroform. On larger scales the solvent was removed in a round-bottomed flask on a rotovap at 50 °C. In small scale preparations the solvent was removed by blowing nitrogen into the sample tube while warming the solution in a water bath. The resulting thin film was dried on a lyophilizer and rehydrated in HBS, for a final lipid concentration of 25 mM. The lipid solution was then passed 10 times through 2 × 100 nm stacked filters using an extrusion device (Northern Lipids Inc., Vancouver, Canada) at 45 °C. Preparation of Micelle-Ovalbumin Conjugates. Ovalbumin was dissolved in HBS at a concentration of 30 mg/mL. An aliquot of from a freshly prepared stock solution of 2-IT in HBS (prepared at a concentration such that the total volume added to the albumin did not exceed 5%) corresponding to 1-5 equiv (depending on the particular experiment) was added to the ovalbumin solution and the mixture stirred at room temperature for 0.5 h. Lipid mixtures were prepared by combining an active lipid (DSPE-MPB or DSPE-ATTA2-MPA) and a micelle-forming lipid (DSPE-ATTA4, OGP, or CHAPS). The lipid was dissolved in 1 part of ethanol, with warming if required, and diluted sequentially with 1 part of HBS, with vortexing and warming, until a total of 9 parts HBS had been added. The total volume should was such that the final concentration of lipid was approximately 10 mM. Any residual solid materials were removed by filtration through 0.22 micron filters. An aliquot of freshly prepared thiolated ovalbumin was added to freshly prepared maleimide-micelles in the appropriate ratio. In most cases this corresponded to 3000 g protein/mol micelle lipid. The micelle-protein mixture was allowed to stir at room temperature overnight. It was then either used immediately without additional purification, or stored at 5 °C for use at a later date. Incubation of Protein-Micelles with Liposomes. Liposomes were mixed with ovalbumin-micelle conjugates in proportions appropriate for the particular experiment. Typically the standard preparation used a 1:19 micelle/liposome lipid ratio, corresponding to an initial protein/final lipid ratio of 150 g/mol when using the typical 3000 g protein/mol micelle lipid reagent. The liposome-micelle mixtures were incubated in a water bath at 60 °C for 30 min and then passed down a Sepharose CL-4B column (0.8 mL of sample/25 mL bed volume) using HBS as the running buffer. Assays. Lipid was assayed by determining the phosphate content as previously described (14). The protein content of the final liposome-protein conjugates was determined using a modified micro-BCA assay as previously described (14), with negative controls being performed where possible to correct for background interference. Protein/lipid ratios in this work are expressed either as g protein/mol micelle-forming lipid, or as g protein/mol final lipid. The first quantity relates to the ratio of protein to lipid present in the initial micelles, while the second quantity refers to the ratio of protein to the total lipid present in the final liposomes. Particle size was measured using a Nicomp 380 submicron particle sizer.

RESULTS The basic design of the process is outlined in Figure 1. The passive conjugation method can be divided into three parts, which can be performed separately. These are (1) preparation of the liposomes, (2) preparation of micelle-protein conjugates, and (3) incubation of the liposomes with the micelle-protein conjugates. This contrasts with the active conjugation method also outlined in Figure 1, where the liposome preparation and


protein derivatization need to be done more or less simultaneously due to the chemical instability of some of the reagents used. The more flexible time constraints associated with the passive conjugation method make it a more attractive choice from the perspective of scalability. This was an important consideration if this technology is to be used in commercial products which are generally prepared in very large quantities. It needs to be kept in mind however that while the “passive conjugation” terminology is being applied to the entire process, the actual conjugation is taking place in the micelle step, whereas the final assembly of the system is mediated through purely physical exchange processes. The apparent coupling efficiency on the micelle surface is implied by the level of protein on the surface of the final liposome composition. It is necessary for the micelles to equilibrate with the liposomes for that to be true. Generally a 1 h incubation is sufficient to equilibrate most systems, but in some cases, for example in liposomes with high levels of securely anchored steric barrier molecules, longer incubation times may be required to equilibrate the system. Presence of Ethanol and Detergents in the Final Formulation. Since the presence of ethanol and/or detergents in the final formulation might affect the integrity of various components during the assembly or after assembly of the final liposome system, it is necessary to ensure that the levels of these materials remain with acceptable limits. Many proteins will denature in the presence of organic solvent. Typically we limit the ethanol content of any solution that proteins might be dissolved in to 5%. In this protocol we mix equal volumes of a thiolated protein solution with a micelle solution. The micelle solution was generally prepared by diluting an ethanolic solution of lipid in buffer. To limit the final ethanol concentration to 5% we needed to prepare the micelles using 1 part of ethanolic lipid solution to 9 parts of buffer solution. The final lipid concentration of the protein-micelle solution was 5 mM. The final ethanol content in the liposome solution can be controlled using the initial liposome concentration. Typically liposomes are prepare with lipid concentrations in the range 10-50 mM. The reference formulation protocol used a 1:19 micelle/liposome lipid ratio, i.e. 10 mM and 50 mM liposome solutions would be treated with 2/19ths and 10/19ths the volume of protein-micelles. This would result in final ethanol concentrations of ∼0.5-1.7%. It might be necessary to use lower initial liposome concentrations if this level of ethanol is a problem in any particular system. The stereotypical micelle formulation in this work was composed of an active lipid and a micelle-forming lipid in a 1:4 ratio and were added to liposomes in a 1:19 lipid ratio. This means that the final active lipid content of the protein-liposome system is 1%, and the final micelle lipid content is 4%. These values are well within the range used in typical “targeted” or “steric barrier” liposome formulations. These ratios were chosen to ensure that this was the case. Thiolation of the Protein. One of the most commonly used methods of preparing protein conjugates involves the use of the maleimide-sulfhydryl coupling reaction. Usually the protein is derivatized with a heterobifunctional cross-linker based either on a maleimide or protected sulfhydryl. This is then reacted with a substrate bearing the complementary reactive function. Most of the literature dealing with liposome-protein conjugation reactions involves modifying the protein with SPDP, deprotecting the sulfhydryl with DTT, and then reacting the product with liposomes containing lipids bearing a maleimide function. Initially we used this chemistry when preparing our micelleprotein conjugates. The use of SPDP presents a number of problems however. The cross-linker generally reacts with the protein in high yield, but the need to deprotect the sulfhydryl with DTT is a major issue, particularly when trying to scale-up the reaction. The DTT treatment is time sensitive, since it may


Figure 2. The effect of increasing the 2-iminothilone/protein ratio on the coupling efficiency. Ovalbumin was thiolated using 5-40 equiv of 2-iminothiolane. The resultant thiolated protein solution was used without workup and incubated with DSPE-ATTA2-MPA/DSPEATTA4 (1:4 ratio) micelles at 3000 g protein/mol micelle lipid. The protein-micelle conjugates were exchanged into POPC/chol (55:45) liposomes at a 1:19 mol/mol ratio by incubation at 60 °C for 1 h. The liposomes were recovered by passing the solution down a sepharose CL-4B column. Final amounts of protein coupled are represented as g protein/mol total lipid.

damage intramolecular disulfide bonds in the protein as well as performing the deprotection of the introduced sulfhydryl group. While this can be controlled to some extent by performing the reaction at lower pH, the reaction needs to be stopped relatively quickly (∼15 min) to prevent significant damage to the disulfide bonds. The reaction is stopped by passing the reaction mixture down a gel column to remove the DTT and exchange the buffer back to pH 7. These constraints make the method unsuitable for large scale preparations. Another disadvantage with the method is that if the DTT is not completely separated from the protein, it may be present in sufficient quantities to quench the reaction with the activated liposomes. A less commonly used thiolating agent is 2-IT. The advantage of using 2-IT is that it is a cyclic reagent that exposes the sulfhydryl group when it reacts with amino functions on proteins. This means that it is not necessary to exchange the buffers or use a specific deprotection reagent, and in principle it may not be necessary to remove excess 2-IT before use of the thiolated protein. 2-IT has been used previously to conjugate proteins to liposomes (6, 7). In those studies it was used in a 20-fold excess, with unreacted reagent being removed by gel filtration before the conjugation of the thiolated protein with liposomes. Since we were interested in minimizing the amount of processing required during the process, we wanted to determine if 2-IT could be used in the passive conjugation protocol without an intermediate gel filtration step to remove unreacted 2-IT. An experiment was conducted where 2-IT was titrated into an ovalbumin solution, and the resultant thiolated product conjugated to DSPE-ATTA2-MPA/DSPE-ATTA4 micelles. The conjugation efficiency was determined by incubation of the ovalbumin-micelles with POPC/chol liposomes at an initial protein/final lipid ratio of 150 g/mol. Unconjugated protein and micelles were removed by gel filtration though sepharose CL4B and the recovered liposome fraction analyzed for lipid and protein content. The results are presented in Figure 2. Interestingly it was found that as the ratio of 2-IT relative to ovalbumin increased, the apparent coupling efficiency decreased. This behavior is likely due to the omission of the gel filtration step for removal of unreacted 2-IT prior to incubation with the maleimide micelles. Hydrolysis of the excess 2-IT in solution probably resulted in quenching of some of the maleimide


functionality, inhibiting the slower protein-micelle conjugation reaction. The best conjugation efficiency was seen at about 5 equiv of 2-IT. Following this experiment we used 5 equiv of 2-IT as our baseline in further optimization studies. Synthesis of Lipids Used. Most of the original proteinliposome conjugation studies reported in the literature used simple lipids derivativatized with a cross-linker to introduce the reactive function. One of the most commonly used lipids is DSPE-MPB. After the development of PEG-lipids as steric barrier molecules in liposomes, some researchers began conjugating proteins to the ends of PEG-lipids in an attempt to get systems with good conjugation efficiencies and favorable pharmacokinetic properties. Maleimide-capped PEG-lipids were good candidates for the first application of micelle-based conjugation techniques in the preparation of liposome-protein conjugates. We were interested in investigating the relationship between the size of the tether in the active lipid and the size of the hydrophilic domain in the micelle-forming lipid in terms of their effects on coupling efficiency. PEG lipids are not ideal for this since they are polymers composed of a wide range of molecular sizes. We have recently developed PEG like molecules based on repeating units of 14-amino-3,6,9,12-tetraoxatetradecanoic acid (ATTA) (13). These ATTA derivatives are suitable for investigating the effects of the relative headgroup size of the active and micelle-forming lipids on the conjugation efficiency since they can be tailored to exact sizes. The synthesis of the ATTA monomer was performed using a modified version of a literature procedure (15). 11-Azido3,6,9-trioxundecanol 1 was prepared by partially mesylating tetra(ethylene glycol), treating the product with sodium azide and recovering the product through a series of selective extraction procedures. Crude 1 was then treated with a suspension of finely powdered potassium bromoacetate and sodium hydroxide pellets in toluene. The literature method used DMF as the solvent; however, we found this to be unsuitable due to decomposition of the solvent under the reaction conditions, which caused considerable problems. The most effective results were obtained with lyophilized potassium bromoacetate. Other methods of preparing the potassium bromoacetate yielded granular forms of the salt, which were found to be relatively ineffective in the reaction. Synthesis of ATTA2 and ATTA4 were performed as previously described (13). The primary active lipid used in this work was DSPEATTA2-MPA. This was prepared by reducing the azido group of ATTA2 to the amine 3 and protecting the amino function using a BOC group to form 4. The carboxyl group was then deprotected by basic hydrolysis to form the acid 5. This was conjugated to DSPE using DCC/NHS chemistry. The BOC protecting group was removed with TFA and the amino function derivatized with MPA using DCC/NHS chemistry. The product 6 was purified by HPLC. Excessive workup of the product should be avoided since this results in hydrolysis and other side reactions with the maleimide group, reducing yields and complicating purification. The storage conditions for the product were important. Over time an unknown material insoluble in organic solvents and water began to form. We believe that this material is a polymer stemming from ring opening of nearby maleimide functions by maleamic acid contaminants. Maleamic acids are much less reactive than maleimides, and excessive presence of those compounds can lead to an apparent loss of activity of the active lipid. Material stored as a lyophilized powder was generally sufficiently stable to be used for up to 4-5 months with noticeable loss of coupling activity. New batches were prepared every 4-6 months. However, material stored as gums formed by evaporating solutions directly using rotovaps usually became unusable within weeks. It is important

Takasaki and Ansell Scheme 1. The Synthesis of DSPE-ATTA2-MPA

that active lipids be stored in a cool dry location once lyophilized and, if significantly below room temperature, be allowed to equilibrate to room temperature to prevent water from condensing on the product. An outline of the synthesis is shown in Scheme 1. It is not practical to synthesize a fresh batch of active lipids prior to each micelle formulation experiment. Consequently any polymerized material formed in the active lipid stocks on storage were removed from the micelles by filtration through 0.22 micron filters. The presence of the impurity can be observed through the appearance of a “haze” in the lipid ethanol and in micelle solution after dilution with buffer. It is necessary to remove this material since it potentially could interfere in size determination measurements. The problem becomes increasingly pronounced as the sample ages. Properly stored reagents were typically effective with minimal insolubles for 4-5 months after synthesis. Material much older than that tended to generate significant amounts of insoluble materials and showed noticeably lower conjugation efficiencies. Whenever using maleimidebased active lipids in formulations of this sort, it is advisable to perform small test runs periodically to ensure that the lipid activity is at an acceptable level. A number of micelle-forming lipids were used in this work. Some of them were commercially obtained but a number of them were based on our ATTAn templates and synthesized in house. These included DSPE-ATTAn, where n ) 1, 2, and 4. The synthesis of these lipids was performed as described previously (16). Effect of Micelle Lipid Structure. The use of micellemediated passive coupling techniques have been reported in a number of publications (6, 7). All of these reports use lipidPEG-maleimide conjugates as the active lipids and PEG-lipids as micelle-forming lipids. There are a number of good reasons for diluting the active lipid with a micelle-forming lipid, even if the active lipid forms


micelles in isolation. First, the active lipid is typically a relatively high cost component in formulations. It is highly unlikely that a large molecule such as a protein can be coupled to every maleimide on pure micelles; therefore, much of the active lipid in those cases is wasted. In principle it should be possible to dilute the active lipid out with another lower cost micelleforming lipid. Second, it should be remembered that maleimide functions are active molecules, which might cause issues in pharmaceutical applications if they are present in high levels. The previously reported work had used DSPE-PEG-2000 as the micelle-forming lipid, and DSPE-PEG-2000-maleimide as the active lipid, in a 4:1 ratio. In that particular case the active group is presented approximately at the surface of the PEG shroud above the micelle; consequently, there should be little inhibition of reaction with proteins. Such systems are expected to exhibit at least some degree of cross-linking since both the micelles and the thiolated protein are multivalent species. Traditionally when doing similar reactions at liposome surfaces, PEG lipids are included to inhibit these cross-linking reactions, based on the principle that, at some ideal concentration range, the PEG is dense enough to inhibit the approach of a liposomeprotein conjugate but not the protein by itself. Since both approaches were being presented as “improvements” over previous technology, we asked the question: what effect does the relative size of the hydrophilic domain of the two lipids present have on coupling efficiency using the micelle based passive conjugation method? To answer this question we designed an experiment where we used micelles composed of a 1:4 ratio of DSPE-ATTA2MPA:micelle-forming lipid, where the micelle-forming lipid was one of the following: DSPE-ATTA1, DSPE-ATTA2, DSPEATTA4, MePEGS-2000-DSPE, and MePEGS-5000-DSPE. The ATTA1, ATTA2, and ATTA4 correspond approximately to PEG 250, 500, and 1000 in size. A micelle-forming lipid was required in this case since the active lipid used, DSPE-ATTA2MPA, did not form a micellar solution by itself. DSPE-ATTA1 was found not to be an effective micelle-forming lipid and was dropped from the study. The four remaining lipid compositions were hydrated by first dissolving the lipids in ethanol and then diluting the solution with nine parts of buffer, such that the final lipid concentration was 10 mM. Previously reported studies on the phase behavior of pure mono methoxy PEG-lipids have shown that MePEG-350-DSPE forms bilayer phases on hydration, whereas the MePEG-750, MePEG-2000, and MePEG-5000 analogues form micellar phases (17). It is likely that DSPE-ATTA1 forms a bilayer phase, while DPSE-ATTA4 forms a micelle phase. It is not immediately clear what form DSPE-ATTA2 adopts, but when fully hydrated it forms a clear solution suggesting that it forms micelles. The MePEGS-5000-DSPE system hydrated very readily, with the other systems showing some signs of nonhydrated material as the size of the micelle-forming lipid headgroup decreased. In the case of the DSPE-ATTA2 system the solution exhibited thermotropic behavior, forming a gel at room temperature which reverted to micellar form on being heated in a 60 °C water bath. The micelles thus formed were incubated with previously thiolated ovalbumin at a protein/micelle lipid ratio of 3000 g/mol. The reaction was allowed to proceed overnight at room temperature. Interestingly, once reacted with the thiolated protein, the DSPE-ATTA2 micelles no longer exhibited the thermotropic behavior previously seen. Each sample was then divided into two parts. One-half was mixed directly with liposomes in a 1:19 ratio and incubated at 60 °C for 30 min. The solution was then run down a sepharose CL-4B column to remove any excess protein or micelles from the sample. The second half of the micelle/protein solutions were


Figure 3. The effect of the micelle-forming lipid headgroup size on coupling efficiency in passive coupling protocols. Ovalbumin was thiolated with 5 equiv of 2-IT and incubated with DSPE-ATTA2-MPA/ micelle-forming lipid (1:4) micelles in a 3000 g/mol ratio. The micelleforming lipids used were as denoted on the figure. The protein-micelle conjugates were incubated with POPC/chol (55:45) liposomes in a 1:19 mol/mol ratio. Panel A: Final bound protein/lipid ratio (g/mol) where the protein-micelle conjugates were filtered before incubation with the liposomes (crossed bars), or added directly to the liposomes without pretreatment (open bars). Panel B: Size increases of liposomes after incubation with the protein-micelle conjugates. The dotted line represents the original size of the liposomes.

filtered through 0.22 micron filters and then treated in the same way as the first half. Protein content was assessed using a modified BCA assay (10) and lipid content with a molybdate assay (10). The samples were also sized with a Nicomp 380 particle sizer. The results of the experiment are shown in Figure 3. The first observation made was that filtering the micelleprotein conjugates prior to incubation with the liposomes had little effect on coupling efficiency except for the DSPE-ATTA2 systems, which showed a marked drop. There was a slight increase in turbidity as the micelle-forming lipid headgroup decreased in size, which was removed by the filtration step. This suggested that cross-linking increased as the headgroup size decreased, which is not a surprising finding. Similar trends were observed with the particle sizing data for the final liposome constructs. Aggregation of the liposomes is indirectly linked to crosslinking in the micelle solutions since it is mediated by proteins that have two or more lipid anchors attached at distal ends. Thus, when one lipid anchor inserts into the liposome membrane, the distal anchor is available to insert into another liposome, inducing aggregation. Multiple distal derivatization of the protein


occurs when a protein conjugated to one micelle surface is able to access and react with another micelle surface. Clearly, increasing the size of the micelle-forming lipid inhibits this cross-linking as evidenced by the particle sizing data. However, increasing headgroup size also inhibits the conjugation reaction, resulting in decreasing conjugation efficiencies. PEG5000 essentially stopped the reaction, suggesting that the maleimide group was inaccessible. Significant inhibition is also seen with PEG2000. ATTA2 and ATTA4 have similar conjugation efficiencies, capping at approximately 50 g/mol, which, as we will show later, corresponds to the saturation of the micelle surface. The apparent coupling efficiency for the filtered DSPEATTA2 micelles is lower than the unfiltered sample due to the removal of aggregated micelle-protein constructs. This in turn is reflected in the sizing data where the protein-liposomes generated from the filtered sample are larger than those from the unfiltered sample. This apparent contradiction is likely due to the removal of the most severely aggregated liposomes during the gel filtration step, resulting in a selective recovery of smaller liposomes. The trend of decreased coupling efficiency as the extent of the steric barrier coverage of the surface of the micelle increases is similar to the behavior seen when conjugating protein to PEGylated liposomes using active coupling methods (18). In that experiment protein is conjugated to the surface of liposomes containing varying amounts of PEG2000 lipids in the membrane. As the level of PEG2000 increases, the conjugation efficiency drops off to a background level at ∼6 mol %. At this point the protein can no longer access the surface due to steric inhibition. The results of these experiments demonstrate that a wide range of micelle compositions are possible provided that the micelle-forming lipid headgroup does not become so large that it prevents access of the protein to the maleimide group. The primary issues to consider in developing optimal reaction/ formulation conditions are aggregation and reaction efficiency. Zero Length Micelle-Forming Lipids. The fact that the passive coupling protocol worked with the DSPE-ATTA2 system posed new questions: How small is it possible for the micelle-forming lipid to be and still comprise a working formulation? How small is it possible for the active lipid to be and still comprise a working formulation? To answer these questions we designed an experiment where we used either DSPE-MPB or DSPE-ATTA2-MPA as the active lipids, and DSPE-ATTA4, OGP, and CHAPS as the micelle-forming lipids, six systems in all. The DSPE-ATTA2-MPA/DSPE-ATTA4 system was used as a reference point. Thiolated protein corresponding to a range of initial protein/final lipid ratios (based on final lipid, after mixture of the protein-micelles to the liposomes) from 50 to 500 g/mol was added to sets of micelle samples from each formulation group (since the final micelle/ liposome lipid ratio is 1:19, this corresponded to protein/micelle lipid ratios of 1000-10000 g/mol micelle lipid). The proteinmicelles (42 in all) were added to liposome samples and treated as described earlier. Lipid, protein, and size analyses were performed for each system. The results for the DSPE-ATTA2MPA/OGP system data set are presented in Figure 4. Results for the other five systems can be seen in the Supporting Information. We used the usual hydration method for preparing the micelles. This involved dissolving the lipid in ethanol and diluting the resultant solution in 9 parts buffer, such that the final concentration was ∼10 mM. This worked well in most cases, except for the CHAPS systems. CHAPS formed a gellike solid in ethanol but formed micelles readily enough when the buffer was added. All systems formed micelles during the hydration process. Two systems showed thermotropic gel

Takasaki and Ansell

Figure 4. The effect of increased protein/micelle lipid ratio. Micelles were composed of DSPE-ATTA2-MPA/OGP (1:4). Ovalbumin was thiolated with 2-IT in a 1:5 ratio and incubated with micelles at various protein/lipid ratios. The initial protein/lipid content is expressed as g ovalbumin/mol total lipid for convenience, the initial protein/micelle lipid (g/mol) ratio is 20× the number shown. Liposomes were based on POPC/chol (55:45), and were treated with protein-micelles in a 1:19 micelle/liposome lipid ratio. Panel A: Protein bound to liposomes as a function of the initial protein content. Panel B: Final liposome size as a function of the initial protein content. The dashed line represents the size of the precursor liposomes.

formation behavior, namely the DSPE-MPB formulation with OGP and CHAPS. Gel formation was heaviest in the latter case. However, both systems could be redissolved into a micellar solution on warming in a 60 °C water bath. It is likely that this behavior is due to transient hydrophobic interactions between the maleimide groups in the absence of some sort of steric barrier molecule on the micelle surface. Addition of thiolated ovalbumin in various ratios resulted in abolition of the thermotropic behavior mentioned above. The reaction was allowed to proceed overnight at room temperature. The systems containing CHAPS at 50 g/mol (initial protein/ final total lipid) showed heavy aggregation, with light aggregation at 100 g/mol (initial protein/final total lipid). No aggregation was observed at higher ratios. Light aggregation was also seen in the DSPE-MPB/OGP system at 50 g/mol (initial protein/final total lipid). Slight turbidity was seen in a number of other formulations at 50 g/mol (initial protein/final total lipid) but could be dispersed on heating. The heavy aggregation observed in some of the systems was likely caused by cross-linking between the micelles, resulting in proteins which have been derivatized with hydrophobic anchors on distal ends of the molecule. Such molecules are undesirable when associated with liposome surfaces due to their ability to cause cross-linking and aggregation of the liposomes. The enhanced aggregation observed in the CHAPS systems at lower initial protein/lipid ratios is probably due to ionic interactions between the CHAPS and protein. At higher initial protein concentrations this effect is no longer seen, most likely as a result of conjugated protein



Figure 5. Mechanism for the inhibition of aggregation. A. Single macro-molecules can access unprotected surfaces readily. B. Surfaces without sufficient density of protein can undergo cross-linking reactions. C. Single macromolecules can access protected surfaces. D. Modified surfaces cannot access other modified surfaces readily, inhibiting cross-linking. E. Increasing concentration of free protein relative to other surfaces increases the rate of reaction of single protein conjugation

acting as a steric barrier molecule on the liposome surface, inhibiting further cross-linking reactions. Figure 4 (and the related Supporting Information) show several interesting trends. In many cases the coupling efficiency increases to somewhere between 50 and 60 g/mol (initial protein/ final total lipid) and then plateaus out. At high ratios it begins to decline. This would suggest that the micelle surfaces are being saturated at some point between 50 and 60 g/mol (initial protein/ final total lipid), and that attempting to achieve higher ratios would reduce coupling efficiency. The particle sizing data (Figure 4 panel B) showed a steady decline in aggregation levels until around 200-300 g/mol (initial protein/final total lipid). We chose 150 g/mol (initial protein/final total lipid) as the standard initial protein/final lipid ratio, since it saturated the micelle surface at reasonable coupling efficiencies with relatively minor size increases due to aggregation. The apparent decline in coupling efficiency seen at high initial protein/final lipid ratios is probably due to the advent of maleimide quenching by excess hydrolyzed 2-IT which is still present in the thiolated protein solution. Another interesting observation was that the saturation level of the DSPE-MPB micelles appeared to be somewhat lower than the DSPE-ATTA2-MPA systems (Supporting Information). This is not surprising since the area available for protein coverage is determined by the radius of the maleimide functionality, which is somewhat larger in the case of DSPEATTA2-MPA than DSPE-MPB. A smaller radius means less reactive surface area and hence a lower saturation level. These results suggest a general model for preparing proteinmicelle conjugates. Both the micelles and the proteins are multifunction entities. Micelles have a great many copies of maleimide on their surface, while the ovalbumin has on average up to five sulfhydryl groups. This means that once a thiolated protein has reacted with the surface of a micelle, it may still be available for reaction to another micelle. Once a protein is conjugated to a micelle however, the probability of it reacting with another micelle is smaller than the free protein alone due to steric hindrance. As the surface concentration increases, the protein starts to act as a steric barrier molecule in its own right and eventually

stops aggregation reactions. In situations where the initial free protein/micelle ratio is large, the rapid coating of the micelles prevents further aggregation reactions. Alternatively, where the initial free protein/micelle ratio is smaller, aggregation can still occur (Figure 5). This suggests that the degree of aggregation in a particular system can be controlled simply by adjusting the initial protein/lipid ratio. There is a limit to how far this can be taken though, since coupling efficiency is likely to be an issue with many proteins, particularly those in limited supply. For most of our work we selected 150 g/mol (initial protein/ final total lipid) as our baseline standard, since aggregation was reasonable and coupling efficiency acceptable (∼35%). If this mechanism is correct, we can predict that changing other parameters should also have effects on coupling efficiency and aggregation. These include the active lipid content of the micelles, initial 2-IT concentration, and the initial protein/final lipid ratio. Effects of Changes to the Active Lipid Content. If the proposed mechanism is correct, we would expect that alterations to the active lipid content should affect the coupling efficiency. A micelle surface concentration of 1000 g/mol (initial protein/micelle lipid) corresponds to 0.022 mol protein/mol lipid. This implies that there is one protein for every 45-50 lipid molecules in the micelle. Our baseline formulation used a micelle composition of active lipid/micelleforming lipid in a 1:4 ratio; in other words there is one protein for every 9-10 active lipid molecules. That would imply that the surface is being saturated, rather than the reactive functions on the surface, since this appears to be the upper limit of conjugation in these systems. We designed an experiment in which the active lipid content in the micelles was changed, but the total amount of micellar lipid relative to liposome lipid was held constant. Thiolated protein was incubated with various samples of micellar lipid in a 3000 g/mol ratio (or 150 g initial protein/mol final lipid) and then treated with POPC/chol liposomes in a 1:19 lipid ratio. The results are shown in Figure 6. As expected reducing the amount of active lipid in the micelles resulted in reduced aggregation. However, coupling efficiency is also adversely affected, falling off as the active lipid content drops.


Figure 6. The effect of reducing the active lipid content while keeping the micelle/liposome lipid ratio constant. Micelles were composed of DSPE-ATTA2-MPA/OGP (1:X). Ovalbumin was thiolated with 2-IT in a 1:5 ratio and incubated with micelles at 3000 g protein/mol micelle lipid. Liposomes were based on POPC/chol (55:45), and were treated with protein-micelles in a 1:19 micelle/liposome lipid ratio, for an initial protein/total lipid content of 150 g/mol. Panel A: Final bound protein content as a function of active lipid content in the micelles. Panel B: Final liposome size. The dashed line represents the size of the precursor liposomes.

A second experiment was done in which the active lipid/ micelle-forming lipid ratio was changed, but with the total amount of micelles added to the liposomes normalized against the active lipid. The final active lipid content was 1%. The results are presented in Figure 7. We had expected that the amount of conjugated protein would increase as the amount of micelle-forming lipid increased, since this should have resulted in more micelles with a corresponding increase in the reaction surface. In fact there were relatively small changes in the conjugation efficiency, with no clear change in particle size. This would imply that either the surface area of the micelles was remaining approximately constant, or that the reaction efficiency on the surface was decreasing as the micelle number increased. The most probable explanation is that both the micelle number and the reactive radius in the context of this experiment are being determined largely by the active lipid. The results suggest that the protocol is relatively insensitive to changes in the micelle lipid composition, at least within the data range covered by the experiment. Effects of 2-IT Ratio on Aggregation. An alternative method for reducing cross linking reactions is to reduce or minimize the number of additional sulfhydryl groups in the thiolated protein. To investigate the effect of the number of cross-linking groups we titrated in 2-IT corresponding to 1-5 equiv per protein, with 5 equiv being our baseline system. The experiment was performed at two initial protein/final lipid ratios, namely 50 and 200 g/mol (initial protein/final total lipid). The results of the experiment are presented in Figure 8. As expected, decreasing the amount of cross-linker used decreased the level

Takasaki and Ansell

Figure 7. The effect of reducing the micelle lipid content while keeping the active lipid/liposome lipid at 1%. Micelles were composed of DSPEATTA2-MPA/OGP (1:X). Ovalbumin was thiolated with 2-IT in a 1:5 ratio and incubated with micelles at 15000 g protein/mol active lipid. Liposomes were based on POPC/chol (55:45) and treated with micelles such that the initial protein/total lipid content was 150 g/mol. Panel A: Final bound protein content. Panel B: Final liposome size. The dashed line represents the size of the precursor liposomes.

of aggregation. Surprisingly, however, reducing the amount of cross-linker used did not dramatically affect coupling efficiency, although a slight drop-off could usually be seen at the lowest cross-linker content, even in the 50 g/mol (initial protein/final total lipid) system. Reducing the Initial Protein Content below the Saturation Level. Since the micelle surface was apparently becoming saturated at a protein/micelle lipid ratio of ∼1000 g/mol, we ran an experiment in which the initial protein content was titrated into micelles below the saturation level. The results are presented in Figure 9 (P/L is represented as initial protein/final lipid). The apparent coupling efficiency in this particular experiment is 6070% for all the data points, except for the final one at 100 g/mol where it has fallen off to 45%. This would indicate that the surface of the micelle is saturated at approximately 1000 g/mol (protein/micelle lipid), which is consistent with the result previously obtained. The limit to the apparent coupling efficiency at 60-70% in this experiment suggests that at least some of the protein is not thiolated, or is thiolated in areas of the protein that are not available for reaction. Modest changes to the sizes of the liposomes were observed after incubation with the protein-micelle conjugates, suggesting that the liposomes were more prone to cross-linking reactions as the protein content decreased. Again, this is to be expected if protein bound on the surface of the micelles acted as a steric barrier, since reducing the protein on the surface should increase the probability of a successful cross-link reaction. Titration of Protein-Micelles into Liposomes. A solution of protein-micelles with an initial protein/micelle lipid ratio of 3000 g/mol was prepared and titrated into liposomes (Figure 10). At this concentration, coupling efficiency is typically 30-



Figure 8. The effect of the 2-IT/ovalbumin ratio. Micelles were composed of DSPE-ATTA2-MPA/OGP (1:4). Ovalbumin was incubated with 1-5 equiv of 2-IT and incubated with micelles at 1000 g/mol or 4000 g/mol. Liposomes were based on POPC/chol (55:45) and incubated with micelles in a 19:1 lipid ratio, so that the initial protein/total lipid content was either 50 or 200 g/mol. Panel A: Final bound protein content as a function of the initial 2-IT/ovalbumin ratio. Panel B: Final liposome size. The dashed line represents the size of the precursor liposomes.

40%. The protein content associated with the final liposome formulations increased in proportion to the amount added. This value tended to fall off at very high levels of added micelles. Figure 10 shows a slight fall off in association efficiency at 300 g/mol (initial protein/final total lipid); however, at this point the ratio of liposome/micelle lipid is 10:1. At these levels the detergent itself may start to affect the system in significant ways. We have titrated the micelles into liposomes to much higher levels (see Supporting Information) and start to see a marked decline in association efficiency, together with a fall in the levels of liposome aggregation at very high micelle contents. Those results suggest that the liposome surfaces might be becoming saturated at those levels, but it is difficult to draw conclusions since at those detergent levels, the surfactants must be playing a role. The levels of aggregation observed in Figure 10 is approximately the same for all the systems studied. This is not surprising since the cross-linking reaction occurs during the micelle-protein incubation, and the same micelle-protein solution was used to prepare all of the final liposome formulations. The systems should show equivalent levels of aggregation provided that the surface of the liposomes was not saturated. Reproducibility of the Conjugation Process. One of the primary problems when using active coupling protocols for preparing protein-liposome conjugates is controlling parameters such as conjugation efficiency. This problem is exacerbated in some cases by the presence of steric barrier molecules on the surface of some liposome formulations, since even small changes in the concentration of these molecules can have significant effects on the accessibility of the liposome surface

Figure 9. The effect of low initial protein/lipid ratios. Ovalbumin was incubated with 5 equiv of 2-IT and added to micelles composed of DSPE-ATTA2-MPA/OGP (1:4) at protein/micelle lipid ratios of 2002000 g/mol. Liposomes based on POPC/chol (55:45) were incubated with micelles in a 19:1 lipid ratio, for initial protein/total lipid ratios of 10-100 g/mol. Panel A: Final bound protein content as a function of initial protein added. Panel B: Final liposome size. The dashed line represents the size of the precursor liposomes.

to the reactive face of the protein. In principle the passive conjugation process should result in far more reproducible formulations since the amount of protein-lipid conjugates formed can be regulated by saturation of the micelle surface. The protein micelles can then be titrated into liposomes at specific concentrations, allowing the user to accurately tailor the particular composition of a protein-liposome formulation. To test this idea we designed an experiment in which three separate sets of protein solutions were thiolated using the same 2-IT stock solution. The three thiolate protein solutions were then incubated with three separately prepared maleimidecontaining micelle solutions. Aliquots of these three solutions were then treated with POPC/chol liposomes as usual. The final micelle-liposome incubation was repeated 4 days later using the same protein-micelle and liposome solutions. After final workup and analysis, the results plotted and presented in Figure 11. The experiment served to demonstrate that relatively reproducible results can be obtained if a standard protocol is followed.

DISCUSSION The primary focus of our laboratory is the development of liposome-based drug delivery systems that are practical candidates for commercialization as pharmaceutical products. Two areas of interest to us have long been the development of a functional “targeted” formulation and artificial vaccines. In both cases, to develop viable products based on these concepts we have considered it important that we have techniques for conjugating proteins of interest to these systems that are both robust and reproducible.


Figure 10. Titration of ovalbumin/micelles conjugates into POPC/ chol (55:45) liposomes. Ovalbumin was incubated with 5 equiv of 2-IT and added to micelles composed of DSPE-ATTA2-MPA/OGP (1:4) in a 3000 g protein/mol micelle lipid ratio. Aliquots of ovalbumin/ micelles were added to liposomes such that the initial protein/total lipid ratio was as indicated in the figure. Panel A: Final bound protein content as a function of the initial protein/lipid ratio. Panel B: Final liposome size. The dashed line represents the size of the precursor liposomes.

Figure 11. Reproducibility of the passive coupling procedure. Three separate ovalbumin solutions were treated with 2-IT in a 1:5 ratio. Aliquots of the thiolated ovalbumin were added independently to three separately prepared micelle solutions (DSPE-ATTA2-MPA/DSPEATTA4; 1:4) at 3000 g protein/mol micelle lipid. Aliquots of the three micelle-ovalbumin conjugate solutions were added to POPC/chol liposomes (55:45; a single stock solution was used) at an initial protein/ total lipid ratio of 150 g/mol. After a few days of storage the micelleovalbumin/liposome incubations were repeated a second time using the same liposome and micelles stock solutions.

Drugs encapsulated in liposomes largely assume the pharmacokinetic characteristics of the liposomes themselves, with the primary variable being the leakage rate of the drug in vivo. This creates limitations in the utility of these systems however, since liposomes distribute in vivo in specific ways. It has long

Takasaki and Ansell

been an objective of many groups in the field to alter this distribution through the attachment of targeting functions on the surface of liposomes, such that they would accumulate in a specific tissue or a specific cell type. Much of this work has focused on the use of proteins such as antibodies to achieve this goal. Many problems have been experienced in these studies however. It is often difficult to determine if a “targeted” liposome has truly reached the target in vivo, since in many cases access to the target requires extravasation to take place first. Extravasation then becomes the rate-limiting step, making it difficult to distinguish between actual targeting and non specific association. In addition, many of the proteins used in these studies have natural effector functions, or are recognized as foreign by the immune system when attached to liposomes. One of the issues faced when comparing different studies or experiments is that very often the levels of conjugated protein vary considerably. This is partly the result of the intrinsic variability in conducting multistep reactions on proteins and partly due to aggregation reactions and/or steric inhibition of reactions on liposome surfaces due to the presence of steric barrier molecules. The development of the work discussed in this paper was motivated to address these issues and provide a conjugation system that was both reproducible and technically simple. The other area of interest to us in this field was the synthesis of artificial vaccines. While working on the development of liposome-encapsulated oligonucleotides (19) we discovered that these formulations had powerful immunostimulatory effects (20). We also made the serendipitous discovery that a subset of these formulations which included nonexchangeable PEG-lipids as part of the lipid composition showed dramatic increases in liposome clearance rates on repeat administrations. This was later shown to be due to an induced immune response to the PEG (21), a molecule which is normally thought of as nonimmunogenic. Following this discovery we were able to show that greatly enhanced immune responses to protein was achieved when they were conjugated to the surface of these oligonucleotide-liposome particles.2 Originally we conjugated proteins to these particles using active coupling methods. It was difficult to get reproducible results with these methods however, necessitating the development of more controllable methods of conjugation. Although the nature of the particle is very different from those used in drug targeting applications, the fundamental issues involved in performing a protein-particle conjugation are much the same. The same techniques should be applicable to both applications. This need led to the development of the general technique described in this paper. Active conjugation methods tend to suffer from a number of serious drawbacks. The most important of these are reaction control, aggregation, and conjugation efficiency. When conjugating a protein to the surface of a liposome, both entities have multiple sites of possible reaction. In the absence of any sort of controlling mechanism, this leads to cross-linking of liposomes and the formation of liposome aggregates. These aggregates are rapidly cleared in vivo and are considered pharmaceutically undesirable. The formation of the aggregates can be suppressed by including steric barrier molecules in the formulation or by controlling the relative concentrations of the reactants. However, these methods lead to the suppression of the reaction through either steric inhibition or by competition 2 Yuan, Z. N., Brodsky, I., Ansell S. M., Klinuk, S.K, and Semple, S. C. (2004) Systemic and mucosal immune responses induced by liposomes with surface-coupled antigen and high levels of encapsulated immunostimulatory oligonucleotides. Unpublished data.


from hydrolysis reactions. This manifests itself through generally low and variable coupling efficiencies. The basic problem with active conjugation is that we are performing two processes simultaneously, namely: (a) associating the protein with the liposome; and (b) forming a lipidprotein conjugate. Since a number of variables affect this process, we end up with a system that is intrinsically difficult to control. The simplest and most obvious solution to the problem is to separate the procedure into two steps, and this is what we do with the passive conjugation method. The lipid protein-conjugate is formed externally from the liposome, after which it is exchanged into the liposome bilayer. In this particular case we are using a micelle as a substitute surface to perform the reaction on. As can be seen from the data in Figure 4, once you reach a certain point it is no longer possible to increase the amount of protein associated with the micelles since the surface has been saturated with protein. This property allows us to prepare a reagent in which the amount of lipid-protein conjugate available for association with the liposomes is determined primarily by the micelle surface area. This can then be titrated into a preformed liposome solution and incubated under appropriate conditions until the proteinlipid conjugates exchanges into the liposome bilayer. The method allows us to make protein-liposome conjugates with a precision not previously possible. Reports in the literature have used similar techniques to prepare protein-liposome conjugates (6, 7). In those cases the active lipid used was a phospholipid-PEG-maleimide derivative. Placing the reactive function on the end of a PEG chain effectively dramatically increases the micellar radius of reaction, meaning that considerably more protein is required to saturate the micelle surface. Since saturation of the micelle surface is advantageous for reproducible results, the active lipids we have used here (DSPE-MPB and DSPE-ATTA2-MPA) should be more effective than PEG-based analogues. The issue of aggregation of the final protein-liposome conjugates is not immediately solved by the approach described here since the micelles can still undergo cross-linking reactions in much the same way liposomes do. However, there are methods for controlling this process. With active coupling protocols this would be achieved by including a steric barrier lipid in the liposome composition. This results in systems where the reaction at a liposome surface with a free protein is more likely than with the much larger protein-liposome conjugate, because of interactions between the steric barrier lipids in the respective liposomes. The same principle applies for micelles, except that in this case the conjugated protein acts as the steric barrier molecule. As the initial protein content increases above the amount needed for saturation, the faster rate of reaction for the free protein would result in increased inhibition of the slower cross-linking reaction and consequent reduction in cross-linking. This is reflected indirectly in the sizing data for the final liposome conjugate. This means that aggregation can be minimized by increasing the initial protein-lipid ratio. The price paid is a lower coupling efficiency. This might be acceptable if the protein is inexpensive or readily available, but in many cases this may not be true and consequently the cost aspect may a factor to consider when designing the specifics of the process used. An alternative approach to the problem is to reduce the probability of crosslinking occurring by reducing the amount of one of the reactive functions. This can be done by reducing the active lipid content in the micelles, or by reducing the level of thiolation in the protein. As can be seen in Figures 6 and 8, both of these approaches work, albeit with lowered coupling efficiency in some cases. The process works by making the cross-linking reaction less likely than the primary conjugation reaction.


In conclusion we have demonstrated that micelle-based passive coupling techniques are highly effective for the preparation of well defined protein-liposome techniques, and that a wide range of micelle-forming and active lipids can be used for this purpose. The ideal system to use would be one in which a relatively large excess of initial protein was used. The active lipid chosen should have a relatively nonexchangeable lipid anchor, such as DSPE, and preferably have a short tether between the lipid anchor and the maleimide function. A micelleforming lipid should be present to dilute out some of the reactive lipid and to facilitate micelle formation where necessary. A range of micelle-forming lipids are suitable; however, the hydrophilic headgroup should not be so large that it inhibits the reaction through steric interactions. The application of these techniques to the preparation of protein-lipid-antisense complexes will be described in a later work. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED (1) Supra, P., and Allen, T. M. (2003) Ligand-targeted liposomal anticancer drugs. Prog. Lipid Res. 42, 439-462. (2) Fortin, A., Shahum, E., Krzysztof, K., and Therien, H. M. (1996) Differential activation of cell-mediated immune functions by encapsulated and surface-linked liposomal antigens. Cell. Immunol. 169, 208-217. (3) Vannier, W. E., and Snyder, S. L, (1988) Antibody responses to liposome-associated antigen. Immunol. Lett. 19, 59-64. (4) Guan, H. H., Budztnski, W., Koganty, R. R., Krantz, M. J., Reddish, M. A., Rogers, J. A., Longenecker, B. M., and Samuel, J. (1998) Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses. Bioconjugate Chem. 9, 451-458. (5) Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press, San Diego. (6) Iden, D. L., and Allen, T. M. (2001) In vitro and in vivo comparison of immunolipososmes made by conventional coupling techniques with those made by a new post-insertion approach. Biochim. Biophys. Acta 1513, 207-216. (7) Supra, P., and Allen, T. M. (2004) Improved outcome when B-cell lymphoma is treated with combinations of immunoliposomal anticancer drugs targeted to both CD19 and CD20 epitopes. Clin Cancer Res. 10, 2530-257. (8) Loughrey, H. C., Wong, K. F., Choi, L. S., Cullis, P. R., and Bally, M. B. (1990) Protein-liposome conjugates with defined size distributions. Biochim. Biophys. Acta 1028, 73-81. (9) Juliano, R. L., and Stamp, L. (1975) The effect of particle size and charge on the clearance rates of liposomes and liposome encapsulated drugs. Biochem. Biophys. Res. Commun. 63, 651-685. (10) Harasym, T. O., Tardo, P., Longman, S. A., Ansell, S. M., Bally, M. B., Cullis, P. R., and Choi, L. S. L. (1995) Poly(ethylene glycol)modified phospholipids prevent aggregation during covalent conjugation of proteins to liposomes. Bioconjugate Chem. 6, 187-194. (11) Blume, G., Cevc, G., Crommelin, M. D., Bakker-Woudenberg, I. A., Kluft, C., and Storm, G. (1993) Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim. Biophys. Acta 1149, 180184. (12) Ishida, T., Iden, D. L., and Allen, T. M. (1999) A combinatorial approach to producing sterically stabilized (Stealth) immunoliposomal drugs. FEBS Lett. 460, 129-133. (13) Dhawan, R., Kadijk, M. G. A., Joikinen, T. J., Feng, M., and Ansell, S. M. (2000) Synthesis of polyamide oligomers based on 14-amino-3, 6, 9, 12-tetraoxatetradecanoic acid. Bioconjugate Chem. 11, 14-21.

450 Bioconjugate Chem., Vol. 17, No. 2, 2006 (14) Ansell, S. M., Tardi, P. G., and Buchkowsky, S. S. (1996) 3-(2Pyridyldithio)propioic acid hydrazide as a cross-linker in the formation of liposome-antibody conjugates. Bioconjugate Chem. 7, 490-496. (15) Schwabacher, A. W., Lane, J. W., Scheisher, M. W., Leigh, K. M., and Johnson, C. W. (1998) Desymmetrization reactions: efficient preparation of unsymmetrical substituted linker molecules. J. Org. Chem. 63, 1727-1729. (16) Ansell, S. M., Kojic, L. D., Hankins, J. S., Sekirov, L. Boey, A., Lee. D. K., Bennett, A. R., Klimuk, S. K., Harasym, T. O., Dos Santos, N., and Semple, S. C. (1999) Application of oligo-(14-amino3, 6, 9, 12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations. Bioconjugate Chem. 10, 653666. (17) Kenworthy, A. K., Simon, S. A., and McIntosh, T. J. (1995) Structure and phase behaviour of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903-1920. (18) Ansell, S. M., Harasym, T. O., Tardi, P. G., Buchkowsky, S. S., Bally, M. B., and Cullis, P. R. (2000) Antibody conjugation methods

Takasaki and Ansell for active targeting of liposomes. In Drug Targeting: Strategies, Principles and Applications (Francis, G. E., and Delgado, C., Eds.) pp 51-68, Chapter 4, Humana Press, Totowa, NJ. (19) Semple, S. C., Klimuk, S. K., Harasym, T. O., Dos Santos, N., Ansell, S. M., Wong, K. F., Maurer, N., Stark, H., Cullis, P. R., Hope, M. J., and Scherrer, P. (2001) Efficient encapsulation of antisense oligonucleotides inlipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochim. Biophys. Acta 1510, 152-166. (20) Mui, B., Raney, D. G., Semple, S. C., and Hope, M. J. (2001) Immune stimulation by a CpG-containing oligodeoxynucleotide enhanced when encapsulated and delivered in lipid particles. J. Pharmacol. Exp. Ther. 298, 1185-1192. (21) Semple, S. C., Harasym, T. O., Clow, K., Ansell, S. M., Klimuk, S. K., and Hope, M. J. (2004) Immunogenicity and rapid clearance of lipososmes containing polyethylene glycol-lipid conjugates and nucleic acid. J. Pharmacol. Exp. Ther. 312, 1020-1026. BC050051R

Micelles as Intermediates in the Preparation of ProteinâLiposome

Recommend Documents

Micelles as Intermediates in the Preparation of ProteinâLiposome