- Email: [email protected]
Liposomes and biopolymers in drug and gene delivery
392
Liposomes and biopolymers in drug and gene delivery Danilo D Lasic* and Demetrios Only the coupling of appropriate
properties
PapahadjopoulosT
of liposomes
and polymers has made possible many of today’s liposome applications,
leading to the first commercially
available
liposomal drug in the USA.
Addresses ‘7512 Birkdale Dr, Newark, CA 94560, USA +Department of Cell and Molecular Biology, Cancer Research Institute. University of California at San Francisco, CA 94113. USA Current Opinion in Solid State & Materials Science 1996. 1:392-400 0 Current Science Ltd ISSN 1359-0286 Abbreviation PEG poly(ethylene)glycol
mal product in the USA. Another important development was the finding that cationic liposome complexes improve gene transfection in vifro [6]. It is now believed that cationic liposomes and polyelectrolytes can effectively condense DNA, which may be a necessary condition for systemic delivery in vjvo and which is being explored in non-viral gene therapy [7-91, (DD Lasic, R Podgornik, H Strey, PM Frederik, unpublished data). This review describes the physico-chemical properties of polymer-coated liposomes and their applications in anti-cancer therapy before considering the interactions of liposomes with free polymers, including cationic liposomes and DNA.
Sterically stabilized Introduction The colloidal state of matter is associated with many special physico-chemical properties which can be exploited in different applications. Liposomes and biopolymers promise many applications in the fields of drug and gene delivery. In some cases, however, neither system has been able to provide all of the desired properties, such as adequate stability or DNA condensation; in these cases it has proved necessary to combine the unique properties of both systems to achieve the desired goal. In addition, for many drugs, the therapeutic window, that is, the difference between the toxic and therapeutic doses, is very narrow. Often, the situation can be improved by changing the pharmacokinetics and biodistribution of drug molecules by using appropriate drug delivery systems. Liposomes, self-closed spherical particles in which selfassembled bilayers encapsulate a volume of the solvent in which they are bathed, are one of the most promising delivery systems [l-3]. In addition, their similarity to cell membranes makes them a very useful model, tool and reagent in biophysics and biochemistry, as well as in several applications, such as a carrier or sustained release system for the microencapsulated (macro)molecules. Given their biocompatibility, biodegradability, low toxicity and immunogenicicy, the main application of liposomes is in drug delivery, especially as an intravenous drug carrier. Early, very optimistic expectations, however, have not materialized, mostly because of their pronounced instability in biological environments, such as in blood circulation. A major breakthrough was the realization that steric stabilization can significantly increase liposome stability and prolong, by several orders of magnitude, their blood circulation times after systemic administration [4]. As a result, sterically stabilized liposomes have yielded substantial improvements in cancer chemotherapy [So], resulting in the registration of the first therapeutical liposo-
liposomes
At the simplest level, it is possible to understand the stability of sterically stabilized systems [lo] as a function of two controlling parameters: polymer chain grafting density, and polymer chain length (degree of polymerization, N)
1111. Corona (i.e. surface layer) of attached polymer causes repulsion (the entropy of chains is reduced upon approach, i.e. loss of configurational entropy) as well as excluded volume and osmotic repulsion. Depending on the size of the polymer(R) and distance between attachment/grafting points (D), the polymer molecules can adopt very different conformations. At very low surface coverages (D>>R), the polymer forms either a pancake-like structure or an inverse droplet-like structure, depending on whether the polymer forms an adsorption or depletion layer on the surface. That is, in the case of weak interaction between polymer and surface the concentration of the polymer is higher at the surface than in the bulk; in the case of weak repulsion the depletion layer is formed in which the surface concentration of polymer is lower than in the bulk. At D> R, the so-called mushroom conformation is present, and when DIR, the polymer chains start to interact, forcing their extension into the so-called brush conformation [ll]. Figure 1 shows these conformations schematically. In this review we shall discuss steric stabilization of colloidal particles by grafting inert polymers onto their surface, and some interactions of colloidal particles with free polymers. Poly(ethylene) glycol (PEG) is by far the most widely used polymer to impart steric stabilization [SO], due to the fact that it is quite soluble in organic solvents and water, and its chemistry is well known (it is a very flexible polymer due to high anisotropy of its monomer i.e. high 1ength:thickness ratio). Herein we shall discuss only
Liposomas and biopolymws in drug and gene delivery Lasic and Papahadjopoulos
Flgura 1
A
L!pld Bilayer
.Polynler
393
[3,4,5*]. Neither the introduction of a larger amount of PEG-lipid .nor lengthening of the polymer chains results in further improved stability in circulation; this is probably attributable to higher lateral pressure of polymer above the surface and increased aqueous solubility of these large molecules. Normally, distearoyl chains are used to increase the anchoring effect, preferably in the mechanically strongest (most cohesive) DSPC/cholesterol bilayers (where DSPC is distearoyl phosphatidyl choline)
[la. Recently it was shown that PEG-lipid can be inserted into preformed liposomes simply by incubation of PEG-lipidfree liposomes with PEG-lipid micelles. For instance, 90 min incubation at T>55’C resulted in almost complete insertion of ZCJooPEG-DSPE from micelles into liposome bilayers [ 171.
YlOLW
A
Schematic presentation of various polymer conformations on the lipcsome surface in the case of PEG with a molecular mass of 2000Da
liposomes, and several mechanisms of liposome coupling to various lipids are shown in Figure 2 [IZ]. PEG can be coupled also on diacyl phosphatidyl glycerol and some other, especially non-ionic, lipids. The generality of polymer stabilization of liposomes was confirmed when workers discovered other polymers which rendered similar biological stability. Poly(Z-methyl2-oxazolidine) and poly(2-ethyl-2-oxazolidine) with N-50 (where N is the degree of polymerization) attached to DSPE (distearoyl phosphatidyl ethanolamine) and incorporated into liposome bilayers at Smol% exhibited a stability profile similar to that of PEG [13]. Poly(acryl) amide and poly(viny1 pyrrolidone) also increased blood circulation times of liposomes. The inferior results, as measured by shorter blood circulation times of liposomes containing these polymers, compared with PEG, are probably attributable to a weak hydrophobic anchor which cause release of these molecules from liposomes [14]. A thorough analysis of the aqueous solubility of PEGlipids, which has pronounced effects on liposome stability, has been performed [15]. As expected, aqueous solubility depends critically on hydrophobic anchors and follows the scaling relationship a NJ/5 with respect to the PEG chain length [15]. Despite its importance, the critical micellar concentration of PEG-lipids has not been reported yet. Preliminary measurements have shown very high values (millimolar range) which are attributed to impurities (DD Lasic, unpublished data). Normally, covalently
liposome bilayers contain 5mol% lipid with attached PEG with a molecular mass of 2000 Da
With the development of monoclonal antibodies and long-circulating liposomes, the targeting of their contents to particular cells become feasible. In addition to classical techniques used to link antibodies, lectins or other ligands to phosphatidylethanolamine, coupling to the far end of the PEG chain has been developed. The inert terminal methoxy group is replaced with a reactive functional group suitable for conjugation after the liposomes have been prepared containing such a reactive polymer-lipid. Antibodies can be coupled to liposomes in several ways (Fig. 3) [18*].
Some experimental
results and discussion
The hypothesis of increased repulsive pressure above membrane and reduced adsorption on the blood circulation lifetimes was tested by measuring the repulsive pressure between membranes with and without incorporated polymer-bearing lipid by using the osmotic stress technique. Figure 4 shows that bilayers containing PEG lipid show much larger interbilayer spacings, and even upon strong compression the bilayers are still spaced 4nm apart compared with surface unmodified bilayers, which show practical collapse to the hard wall (Born) repulsion [ 19,201. The interbilayer repulsion can be calculated from the repulsive pressure of surface-attached polymer in a mushroom configuration according to:
P - (5/2) kTNlDz/a (a/(A/2))w3 where N-44, size of the monomer a -0.35 nm, and distance between grafting points D==3.57nm; k is Boltzmann constant and T is temperature. The value obtained is in good agreement with experimental data [19]. For distances A>& (where A, is the thickness of the polymer layer), the repulsive pressure is zero. Theoretical extension of this simple law leads to the parabolic decay instead of steep single-step decay at /I>&. The sensitivity of these results, however, cannot distinguish between
394
Biomaterials
Figure 2
0
mPEG-0 +
N-PE H
0
I
%-PEG
mPEGdichlorotxiazine mPEG
Rl
/d
K-PEG p+
mPEG-0
S:fPEG
N-PE H
PE
mPEG-tresylate i rnPEG,
,PE z
1
Several different reaction, pathways for the conjugation of PEG to diacyl phosphatidylethanolamines.
The linkage obtained
by tresylate reaction
(PEG-NH-), as shown on the bottom and cited in all available literature, will have to be revised in the light of a recent finding [56] relatmg to the PEG-O-OSO-HCH-CO-NHbond (S Zalipsky, personal commumcation), which is considerably less stable than the originally proposed secondary
amine linkage. (Figure courtesy of S Zalipsky.)
parabolic or step decay, indicating that the simple scaling approximation is rather good [ZO]. Similar data (Fig. 4) have also been obtained using the surface force apparatus [ZO]. These results also show increased repulsion with increasing amount of PEG polymer. Surface force measurements have found reversible repulsive force at all separations, and the thickness of the steric barrier was found to be controlled by the amount of added PEG-lipid [ZO].
where RR is the radius of gyration of the polymer in a theta solvent and corresponds to the thickness of extending polymer and can be substituted by Rb. (Flory radius=nf@lj) in good solvents. Despite an underestimation of the polymer layer thickness in the low coverage regimen, both models are able to fit the force-distance profiles rather well (201, as shown on Figure 5. Scaling analysis fit, which also describes the measured dependence rather satisfactorily, can be expressed as: F,(//)/R = 1.6 (2 kT/Dz) [(R,:/h)s/J -11
At low coverages (dilute mushroom), Dolan and Edwards mean-field theory of steric forces and scaling model described experimental data satisfactorily. At higher coverages, Alexander-deGennes theory based on scaling concepts described the data better, and a more complex hlilner-Witten-Cates mean-field treatment [21] did not improve the fit (201. At low coverages and in the mushroom model, the Dolan Edwards mean field expression for force between two curved cylindrical surfaces of radius K can be described by:
At higher grafting densities, the force could be described
FJh)/R = 72 (kTID2) exp (--h/R,)
n, = D (R,:lD).Y~
where the numerical
prefactor
is close to expected
unity
[201. that is, in the brush regime, by:
F,(h)/R = (16 kTn /I,)/ (3503) [ 7(2/i,/b)~~~ + 5 (h/2/1,)7/4 -121 where
Liposomes and biopolymers in dnrg and Qene deliiwn
lasti and Papahadjopoulos
395
Figure 3
Biotia-DOPE AbBiotin
+
Avidin
+
A&SH
Ab-Biotin-Avidin-Biotia-Liporomer
p
Biotinyhtcd@somcs
Biotinyhtedantibody
MPB-DOPE
Biotio-Liporomcs
Immlmoli~
0
+
PDP-DOPE
::
R
S-S-fCI-&-C-NH-DOPE-Liposomc
S-(CH,h-C-NH-DOPbLiposomer MPB-antibody
PDP-PE Ii-
Hz-PEG-DSPE
Ab-CHO
+
f:
NH~NH-C-CH2-NH-C-O-[(CH~~-O]~-C-NH-DSPE-Liposomea
Oxidizedantibody
Hydmzidc-PEG-PEliposunes 1 :: :: :: Ab-CH+4NH--C--CH2-NH-C-O-[(CH2~-O]a-C-NH-DSPE-Liposomes Imfnd~
PDP-PEG-DSPE
::
%
S-S--fCH,),-C-NH--_I(CH2~O].-C-NH-DSPE-Liposomes
MPB-8lltibOdy
PDP-PEGPE liposomcs
P
::
S-(CHzh-C-NH-_[(CH&O].-C-NH-DSPE-Lipoaomcr
Several different reaction schemes for the attachment of antibodiis onto liposomes. Abbreviation are as follows: Ab is antibody, DOPE is dioleoyl phoaphatidylethanolamine, MPB is (maleimidophenyl)butyroyl, PDP is (pyridylithio)propionoyl, Hz is hydrazide, PEG is poly(ethylene) glycol and PE is phosphatidylethanolamine. (Published with permission from 1161.)
The force between two cylindrical surfaces (F,) and repulsive pressure (P), as measured by the osmotic
stress technique, approximation:
can be calculated
using the Derjaguin
Liposomes and biopolymen
Figure 5
in drug and gene dolivery Lasic and Papahadjopoulos
397
The first therapeutic studies were performed with epirubicinand doxorubicin-containing liposomes. The results obtained were very encouraging: whereas treatment with free drug and drug encapsulated in conventional liposomes did not reduce tumor growth, treatment with the drug administered in sterically stabilized liposomes caused complete remission of the implanted tumors (281. These studies, which were performed in mice models of colon carcinoma and mammary tumors, yielded complete remission of slowly growing tumors and of the cytostatic effect of quickly growing tumors, and were followed by reports of effective suppression of the growth of human xenografts in nude mice [SO]. These data have been reviewed [29-331.
The explanation for this effect is simple: tumor tissue is often characterized by badly formed and leaky vasculature. Small particles, which can circulate for prolonged periods of time, therefore can accumulate in sites where the blood vessels allow extravasation. Indeed, studies of tumor accumulation of drug encapsulated in sterically stabilized liposomes have shown that up to 10% of an injected dose appears in the neoplastic tissue after systemic administration. Obviously, this passive targeting concept can be used in the treatment of other diseases, such as inflammations and infections in which the body itself increases vascular permeability to allow influx of repair cells and efflux of damaged material [34,35].
I
I
I
I
I
Successful pre-clinical efficacy studies have been reproduced in humans, and before the commercial availability in late 1995, more than 1500 patients with Kaposi sarcoma were treated with doxorubicin encapsulated in sterically stabilized liposomes. This preparation eventually became the first liposomal therapeutic to be approved by the USA FDA (Federal Drug Administration) in November 1995. The formulation is now being tested in several other tumor models.
8
9.0% =PEG-DSPE
_A-dG
Although we have concentrated mostly on liposome stabilization, surface-decorated liposomes can serve a variety of other purposes. The conjugation of antibodies, lectins, oligosaccharides and other (macro)molecules afford liposomes specific reactivity with specific molecules, polymers, surfaces or cells and appropriate receptors on their surface [3].
11
g,o_/ , ,MwcJyo;,o ,. 1 6
8
10
12
14
16
18
Distance, D (nm)
Theoretical fit of force-distance profiles obtained by surface force apparatus. The Dolan and Edwards (D&E), deGennes (dG) and Milner-Witten-Cates (MWC) models are used. (Adapted from [201.)
Earlier attempts at antibody-directed targeting of liposomes [36-391 had severe limitations with regard to applications in who because of their rapid non-specific uptake by the cells of the reticuloendothelial system. It was therefore important to establish whether steric stabilization could be used in conjunction with surface ligands such as antibodies to achieve cell-specific targeting in vivo.
398
Biomaterials
An early observation with sterically stabilized liposomes was that their accumulation in implanted mouse tumors was further increased when tumor-recognizing antibodies were conjugated to the liposome surface [40]. The ability to target these liposomes to other specific tissues by specific ligands was clearly demonstrated using antibody-recognizing lung endothelial cells [41]. Recent studies from several laboratories have shown that sterically stabilized liposomes bearing antibodies to specific cells do not lose the ability of long circulation times in blood and, additionally, anti-tumor activity, if injected at an early stage in tumor development [42,43]. This is quite encouraging with regard to the future possibilities of targeting in vtio, and is of obvious significance in tissues where these liposomes can penetrate to a certain extent the target cells, such as liver, spleen, areas of inflammation and possibly in some tumors. Immunogenicity of these liposomes (i.e. their clearance from blood after repeated administrations), however, has not been studied yet.
Although sterically stabilized liposomes are known to localize within implanted tumors beyond the endothelial layer [44,45], it is not possible to predict whether liganddirected targeting will greatly enhance their accumulation and/or their efficacy in solid tumors. This is because solid tumors do not have an efficient drainage system and allow only limited diffusion of large macromolecular assemtjlies such as liposomes [45,46]. Any extravasated particles, therefore, are not expected to have access to the majority of tumor cells for directed delivery of their contents. In this context, the release of small diffusible drug molecules from the liposome interior would be important for the delivery of cytotoxic material to other proximal tumor cells in the surrounding area. Some approaches to programmable release of the encapsulated agents, which rely on direct or indirect polymer-liposome interaction, are described below. Data from therapeutic experiments demonstrating increased antitumor efficacy against mouse tumors not sensitive to the free drug have confirmed this hypothesis and have provided the basis for clinical trials [28,43,47-SO].
In addition to stabilization and targeting, surface active groups can induce vesicle aggregation, and fusion among themselves or with cells, and can control leakage and possibly phase behavior. Inclusion of lipids with timedependent conjugated charge or polymer can generate additional ways of regulating colloidal and mechanical liposome stability and phase behavior. For example, bilayers composed of micelle- and inverse-micelle-forming molecules, such as PEG-lipids and dioleoyl phosphatidylethanolamine, form stable bilayers. However, if the PEG polymer is cleaved-by hydrolysis of a boad with known reaction kinetics, by photo-, heat-,
or pH-induced trigger, or by simply the dissociation of the whole molecule from the bilayer because of shorter acyl chain(s), for example-phase transition occurs and encapsulated material is released (S Zalipsky, D Papahadjopoulos and collaborators, unpublished data). In addition to lamellar-hexagonal phase transition, one can induce lamellar-micellar transition by cleaving acyl chains of the lipids. Fusion or liposome dissolution (lamellar-micellar phase transition) can be induced also by free polymers which change their conformation as a function of pH [51,X].
PEG-lipids titrated into lipid bilayers have also shown some other applications, such as in the formation of stable emulsions [53-551. The observation that such bilayers can generate extremely stable foam [29] has not been investigated further. Two other cases of liposome interactions with non-conjugated polymers can be mentioned: the ability of free PEG polymers to induce liposome fusion; and polyelectrolyte-oppositively charged liposome interaction.
The most rapidly developing field in the applications of liposomes and other colloidal and macromolecular particles is their role in the delivery of DNA plasmids or short single-strand oligonucleotides with antisense sequences into cells. Mostly cationic liposomes and some positively charged polyelectrolytes, or their combinations, are used to condense and deliver DNA into cells. The structures of cationic lipid-DNA complexes are not yet understood. Because of unwanted toxic effects and lack of specificity and stability, we believe that cationic liposome-DNA complexes are only an intermediate solution. and in the long run these complexes will be coated with anionic lipids and possibly surface-grafted polymers as well as targeting ligands. Some other polycations, cationic polypeptides or polyelectrolytes will find use in DNA condensation and enhancement of encapsulation in anionic liposomes as well as some macromolecules to interfere with endosomal disintegration of endocytosed material or induce direct fusion. Therefore, in this area, surface modification and biocompatible polymers will represent a major development of such ‘artificial viruses’. It is hoped that the interplay of colloid and polymer science coupled with organic synthesis will lead to rational design of ‘intelligent’ colloidal systems and novel applications.
Undoubtedly, phase, colloidal, and biological stability and interaction characteristics of liposomes can be further improved and tailored by coupling appropriate macromolecules, possibly with time-dependent membrane association kinetics, onto their surfaces. We believe that such improvements will lead to novel drug and gene delivery systems.
Liposomes end biopolymers in drug and gene delivery Lasic and Papahadjopoulos
References
and recommended
reading
23.
l.
Kenworthy AK, Simon SA, Macintosh TJ: Structura and phase
behavior of lipid suspensions contalnlng phospholipids with covalently attached PEG Biophys I 1 QQ5,68:1903-1920.
Papars of particular interest, published within the annual period of review, have been highlighted as: .
399
24.
Kenworthy AK, Hristova K, Needhsm D, Macintosh TJ: Range and magnitude of sterlc sterlc pressure between bilayers containing phospholiplds with covakntly atfached PEG Biophys J 1995, 68:1920.
of special interest of outstanding interest
1.
Banghem AD, Hill MW, Miller NGA: Preparation and use of liposomes as models of biological membranes. Methods Membr BiollQ73, 1 :I -68.
25.
Lssic DD, WoodIe MC, Martin FJ, Valentincic T: Phase diagram study of ‘Staalth’-lipid tactthin mixtures. Period Bill 1981, 27:28?-281.
2.
Papehadjopoutos D (Ed): Liposornes and their use in biology. Ann NY Acsd Sci 1870, 378:1-412.
26.
Woodte MC, Cotlins LR, Sponsler E, Kossovski N, Pspahadjopoulos D, Martin FJ: Sfarlully stabtltzad liposomes:
3.
Lasic DD: Liposomes: from Physics to Applications. Amsterdam: Elsevier; 1QQ3:1-575.
4.
Papshadjopoulos D, Allen TM, Gsbizon A, Mayhew E, Hu~I~~Q SK. Woodle MC. Lssii DD. Redemenn C. Martin FJ: Stertcallv stablIlted Ilpo&mes: improvements in ‘pharmacokinetics &d antttumor tharapautk efficacy. Proc Nat/ Acad Sci USA 1991, 88:11460-l 1464.
Lesic DD, Martin F (Eds): Stealth Liposomes. CRC Press: Bocs 5. . Raton; 1 QQ5:1-289. Reviews the physics, chemistry, biology, phannscology and rnedii applications of stericslly stabitiied liposomes. 6.
Felgner PL, Gadek TR, Holm M, Roman R, Wenz M, Northrop J, Ringold M, Dsnielsen H: Lipofactton: a highly effictent lipidmediated DNA transfectton process. Proc /Vat/ Acad Sci USA 198?,84:?413-7417.
7
Lssic DD, Bsrenholz Y (Eds): Liposomes: From Gene Delivery and Diagnostics to Ecology. Boca Raton: CRC Press; 1 QQ6:1-389.
8.
Minsky A, Ghirlando R, Gems& H: Structural features of DNA-c&tonic liposome complexes. In Liposomes: from Gene Delivery and Diagnostics to Ecology. Edited by Lssic DD, Berenholz Y. Bocs Raton: CRC Press; 19QS:?-30.
Q.
Gao X, Huang L: Potentration of &tonic mediated gene delivery by polycattons. 35:1027-l 036.
liposomeBiochemistry 1996,
Lssic DD, Gabiion A, Huang K, Martin FJ, Pspshad$poulos
D:
Sterically stabilized liposomes: a hypothesis on the molecular origin of extended circulation timas. Biochim Biophys Acta IQQI,
1070:18?-192.
28.
Husng SK, Mayhew E, Giteni S, Lasic DD, Martin FJ, Pspshadjopoub D: Pharmacokinattcs and thampautks of sterkalfy stabiltzed liposomas In mtca with C-26 colon carcinoma Center Res 1982, 52:6??4-6781.
29.
Lssic DD: Starkally stabillzad 1994,33:1685-l 699.
30.
Paoehsdiilos D: Stealth lloosomes: from stark stablltxatlon to ‘farg&g. In Liposomes: F&m Gene Delivery and Diagnostics to Ecology. Edited by Lasic DD, Barenholz Y. Boca Rston: CRC Press; 1 Q96:1-6.
31.
Woodle MC, Lssic DD: Stertcally stabillzad Siophys Acta 19Q2, 1113:1?1-1 QQ.
32.
Working P, Newman M, HU~~IQ SK, Vsage J, Mayhew E, LaGi DD:
vesiclas
Angew Chem lnt Ed Eng
liposomes.
Biochim
Pharmacokinetlcs, biodlstribution and therapeutic efficacy of doxorubicin encapsulated in stealth liposomas. J Liposome Res 1994, 41667-682.
34.
Dewhirst D. Needhem D: Gttravasatton of steatth Iloosomes into tumom. In Liposomes: From Gene Delivery and~l)&nosfics to Ecology. Edited by Lasic DD, Bsrenhotz Y. Bocs Raton: CRC Press; 1 QQ6:12?-138.
35.
Huano SK. Martin FJ. Friend DS. Psoshsdiitos D: Mechanism of stialth ‘llposome’rccumulatkr~ in so& pathologkal ttssues. In Liposomes: From Gene Delivery and Diagnostics to fco/ogy. Edited by Lssic DD, Bsrenhob Y. Bocs Raton: CRC Press; 1996:ll Q-l 26.
36.
Heath TD, Fraley R, Papshadjopoulos D: Antibody targetfng of liposomes: call spectftclty obfalned by conjugatton of (Fab’), to vesicle surface. Science 1980, 210:539-541.
37.
Silvius JR, Zuckermann MJ: lnterbilayer transfer of phospholipid anchored macromolecules via monomer diffusion. Biochemistry 1993, 32:3153-3159.
Wolff B. Greooriadis G: The use of monoclonal anttbodv Thy1 gdl for”the targeting of liposomas to AKR-A cells in vftro and in vfvo. Biochim Siophys Acta 1984, 802:259-266.
38.
Hansen EC, Kso GY, Masse E, Allen TM, Zstipsky S: Attachment of anttbodtes to sterkally stabilized liposomes: evaluatton, comparison and optimization of coupling procedures. B&him Biophys Acta 1995, 1239:133-l 44.
Leserman LD, Barbet J. Kouritsky FM, Weinstein JN: Targeting to cells of fluorescent llposomes covalengy coupled with monoclonal antibody of protein A. Nature 1980, 288602-604.
39.
Huang A, Huang L, Kennel SJ: Monoclonal antibody covalently coupled with fatty acid. J Biol Chem 1980, 255:8015-8020.
40.
Pspshadjopoulos D, Gab&n A: Targeting of liposomes cells in vfvo. Ann NY Acad Sci 1987, 507:64-71.
41.
Maruysma K. Kennel, S, Huang L: Lipid composttion is important for highly effident target binding and retention of immunoliposomes. Proc Nat/ Acad Sci USA 1990, 8715744-5747.
42.
Ahmad I, Longenecker M, Samuel J, Allen TM: Antibody targeted delivery of doxorublcln in stealth liposomes can eradicate lung cancer in mice. Cancer Res 1993, 55:1484-l 487
43.
Park JW, Hong K, Carter P, Asgari H, Guo LY, Keller GA, Wirth C, Shstaby R, Knotts C, Wood WI, Pspshadjopoulos D: Development of anti pl85Hgss immunoliposomes for cancer therapy. Proc Nat/ Acad Sci USA 1995, 92:1327-l 331.
44.
Huang SK, Lee K, Hong K, Friend DS, Papshadjopoulos D: Microscopic localixation of sterkally stabilized llposomes in colon carcinoma-bearing mice. Cancer Res 1992, 52:5135-5139.
11.
De Gennes PG: Polymers at intarface. Sci 1987, 27:18Q-209.
12.
Zalipsky S: Polyethylene glycol-lipid conjugates. In Liposomes: From Gene Delivery and Diagnostics to Ecology. Edited by Lssic DD, Berenholz Y. Boca Raton: CRC Press; 1 QQ6:Q3-102.
13.
Woodle MC, Engben CM, Zslipsky S: New amphiphatk polymer-lipid conjugates forming long-clrculattng RBS evadlng liposomas. Bioconjug Chem 1 Q94, 5:493-496.
14.
Torchilin VP, Shtilmsn MI, Trubetskoy VS, Whitemsn K, Milstein AM: Amphiphilic vinyl polymars effactively prolong liposome circulation time in vfvo. Biochim Biophys Acta 1994, 1195:181-184.
17
27
Atlen TM: Long-circulating starkally stabllizad liposomes for targeted drug delivery. Trends Pharma Sci 1994, 15:215-220.
Napper DH: Polymer Stabilization of Colloidal Dispersions. New York: Academic Press; 1983.
16.
of electrophorettc moblllty but not surfaca potential.
Biophys J 1 QQ2,61:902-907.
33.
IO.
15.
re&cU&
Adv Colloid interface
PS Uster, TM Allen, BE Daniel, CJ Mender, MS Newman, GZ Zhu: FEBS Lett 1996, in press.
18. Lssic DD, Needhsrn D: Stealth ltposomes - a prototypical . biomaterial. Chem Rev 1995, 95:1601-l 628. A review of the mechanical and sterical properties of liposomes. 19.
Needhsm D, Hristova K, Macintosh TJ, Dewhirst D, Wu N, Lssic DD: Polymer grafted liposomes: physical basis for the stealth property. J Liposome Res 1992, 2:41 l-439.
20.
Kuhl T. Leckbsnd D . L&c DD. lsraelechvili JN: Modulatton of Interaction forces b&en bilayers exposing short chained ethylene oxide head groups. Biophys J 1984, 66:1479-l 488.
21.
Milner S: Polymer brushes.
22.
Baekrnark TR, Elsnder G, Lssic DD, Sackmann E: Conformational transitions In monolayers of phospholipld-polyethyteneoxtde lipo-polymers and interaction forces wtth solid surfaces. Langmuir 1995, 11:39?5-3987.
Science
1991, 251:205-210.
to tumor
400
Biomateriais
45.
Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D. Jain RK: Microvascular permeability and interstitial penetration of stericaiiy stabilized iiposomes in a human tumor xenograph. Cancer Res 1994, 54:3352-3359.
51.
Thomas U, Tirrell DA: Poiyeiectroiyta sensitized vesicles. Act Chem Res 1992, 25~336-342.
52.
46.
Wu NZ, Da D, Rudolf TL, Naadham D, Dewhirst M: increased microvascular permeability contributes to preferential accumulation of stealth ilposomes in tumor tissue. Cancer Res 1993,53:3?65-3770.
Parente RA, Nir S, Szoka FC: Mechanism of leakage of phosphoiipid vesicles induced by the peptide GALA Biochemistry 1990, 29:1720-l 728.
53.
47.
Mayhew E, Lasic DD, Babbar S, Martin FJ: Pharmacokinetics and antitumor activity of epirubicin encapsulated in iongcirculating iiposomes. Int J Cancer 1992, 51:302-309.
Wheeler JJ, Wong K, Ansall SM, Masin D, Baily MB: Polyethylene giycoi modified phosphoiipids stabilize emulsions prepared from triacyigiyceroi. J Pharm Sci 1994, 83:1558-l 564.
54.
48.
Vaage J, Mayhew E, Lasic DD, Martin FJ: Therapy of primary and metastatic mouse mammary carcinoma by doxorublcin encapsulated in stericaiiy stabilized iiposomes. Int J Cancer 1992, 51~942-947.
La&c DD: Afterword and conclusion. In Stealth Lioosomes. Edited by Lasic DD, Martin F. Boca Raton: CRC Press; 1995:279-280.
55.
Warriner HE, idziak SH, Slack NL, Davidson P, Safinya CR: Lameiiar biogeis: fluid membrane based hydrogeis containing polymer lipids. Science 1996, 271:969-973.
56.
Gais HJ, Ruppart S: Modification and immobilization of protelns with PEG tresyiate and poiysaccharide tresyiates: evidence suggesting a revision of the coupling mechanism end the structure of the polymer-polymer linkage. Tetrahedron Lett 1995,36:2837-2838
49.
50.
Williams SS, Aiosco TR, Mayhew E, Lasic DD, Martin FJ, Banked RB: Arrest of human lung tumor xenograft growth in SCID mice using doxorubicln encapsulated in stericaliy stabilized iiposomes. Cancer Res 1993,53:3954-3958. Allen TM, Mehra T, Hansen C, Chin YC: Stealth liposomes: an improved sustained reiese system for arabinofuranosyicytosine. Cancer Res 1992, 52:2431-2436.
phosphoilpid
Liposomes and biopolymers in drug and gene delivery Danilo D Lasic* and Demetrios Only the coupling of appropriate
properties
PapahadjopoulosT
of liposomes
and polymers has made possible many of today’s liposome applications,
leading to the first commercially
available
liposomal drug in the USA.
Addresses ‘7512 Birkdale Dr, Newark, CA 94560, USA +Department of Cell and Molecular Biology, Cancer Research Institute. University of California at San Francisco, CA 94113. USA Current Opinion in Solid State & Materials Science 1996. 1:392-400 0 Current Science Ltd ISSN 1359-0286 Abbreviation PEG poly(ethylene)glycol
mal product in the USA. Another important development was the finding that cationic liposome complexes improve gene transfection in vifro [6]. It is now believed that cationic liposomes and polyelectrolytes can effectively condense DNA, which may be a necessary condition for systemic delivery in vjvo and which is being explored in non-viral gene therapy [7-91, (DD Lasic, R Podgornik, H Strey, PM Frederik, unpublished data). This review describes the physico-chemical properties of polymer-coated liposomes and their applications in anti-cancer therapy before considering the interactions of liposomes with free polymers, including cationic liposomes and DNA.
Sterically stabilized Introduction The colloidal state of matter is associated with many special physico-chemical properties which can be exploited in different applications. Liposomes and biopolymers promise many applications in the fields of drug and gene delivery. In some cases, however, neither system has been able to provide all of the desired properties, such as adequate stability or DNA condensation; in these cases it has proved necessary to combine the unique properties of both systems to achieve the desired goal. In addition, for many drugs, the therapeutic window, that is, the difference between the toxic and therapeutic doses, is very narrow. Often, the situation can be improved by changing the pharmacokinetics and biodistribution of drug molecules by using appropriate drug delivery systems. Liposomes, self-closed spherical particles in which selfassembled bilayers encapsulate a volume of the solvent in which they are bathed, are one of the most promising delivery systems [l-3]. In addition, their similarity to cell membranes makes them a very useful model, tool and reagent in biophysics and biochemistry, as well as in several applications, such as a carrier or sustained release system for the microencapsulated (macro)molecules. Given their biocompatibility, biodegradability, low toxicity and immunogenicicy, the main application of liposomes is in drug delivery, especially as an intravenous drug carrier. Early, very optimistic expectations, however, have not materialized, mostly because of their pronounced instability in biological environments, such as in blood circulation. A major breakthrough was the realization that steric stabilization can significantly increase liposome stability and prolong, by several orders of magnitude, their blood circulation times after systemic administration [4]. As a result, sterically stabilized liposomes have yielded substantial improvements in cancer chemotherapy [So], resulting in the registration of the first therapeutical liposo-
liposomes
At the simplest level, it is possible to understand the stability of sterically stabilized systems [lo] as a function of two controlling parameters: polymer chain grafting density, and polymer chain length (degree of polymerization, N)
1111. Corona (i.e. surface layer) of attached polymer causes repulsion (the entropy of chains is reduced upon approach, i.e. loss of configurational entropy) as well as excluded volume and osmotic repulsion. Depending on the size of the polymer(R) and distance between attachment/grafting points (D), the polymer molecules can adopt very different conformations. At very low surface coverages (D>>R), the polymer forms either a pancake-like structure or an inverse droplet-like structure, depending on whether the polymer forms an adsorption or depletion layer on the surface. That is, in the case of weak interaction between polymer and surface the concentration of the polymer is higher at the surface than in the bulk; in the case of weak repulsion the depletion layer is formed in which the surface concentration of polymer is lower than in the bulk. At D> R, the so-called mushroom conformation is present, and when DIR, the polymer chains start to interact, forcing their extension into the so-called brush conformation [ll]. Figure 1 shows these conformations schematically. In this review we shall discuss steric stabilization of colloidal particles by grafting inert polymers onto their surface, and some interactions of colloidal particles with free polymers. Poly(ethylene) glycol (PEG) is by far the most widely used polymer to impart steric stabilization [SO], due to the fact that it is quite soluble in organic solvents and water, and its chemistry is well known (it is a very flexible polymer due to high anisotropy of its monomer i.e. high 1ength:thickness ratio). Herein we shall discuss only
Liposomas and biopolymws in drug and gene delivery Lasic and Papahadjopoulos
Flgura 1
A
L!pld Bilayer
.Polynler
393
[3,4,5*]. Neither the introduction of a larger amount of PEG-lipid .nor lengthening of the polymer chains results in further improved stability in circulation; this is probably attributable to higher lateral pressure of polymer above the surface and increased aqueous solubility of these large molecules. Normally, distearoyl chains are used to increase the anchoring effect, preferably in the mechanically strongest (most cohesive) DSPC/cholesterol bilayers (where DSPC is distearoyl phosphatidyl choline)
[la. Recently it was shown that PEG-lipid can be inserted into preformed liposomes simply by incubation of PEG-lipidfree liposomes with PEG-lipid micelles. For instance, 90 min incubation at T>55’C resulted in almost complete insertion of ZCJooPEG-DSPE from micelles into liposome bilayers [ 171.
YlOLW
A
Schematic presentation of various polymer conformations on the lipcsome surface in the case of PEG with a molecular mass of 2000Da
liposomes, and several mechanisms of liposome coupling to various lipids are shown in Figure 2 [IZ]. PEG can be coupled also on diacyl phosphatidyl glycerol and some other, especially non-ionic, lipids. The generality of polymer stabilization of liposomes was confirmed when workers discovered other polymers which rendered similar biological stability. Poly(Z-methyl2-oxazolidine) and poly(2-ethyl-2-oxazolidine) with N-50 (where N is the degree of polymerization) attached to DSPE (distearoyl phosphatidyl ethanolamine) and incorporated into liposome bilayers at Smol% exhibited a stability profile similar to that of PEG [13]. Poly(acryl) amide and poly(viny1 pyrrolidone) also increased blood circulation times of liposomes. The inferior results, as measured by shorter blood circulation times of liposomes containing these polymers, compared with PEG, are probably attributable to a weak hydrophobic anchor which cause release of these molecules from liposomes [14]. A thorough analysis of the aqueous solubility of PEGlipids, which has pronounced effects on liposome stability, has been performed [15]. As expected, aqueous solubility depends critically on hydrophobic anchors and follows the scaling relationship a NJ/5 with respect to the PEG chain length [15]. Despite its importance, the critical micellar concentration of PEG-lipids has not been reported yet. Preliminary measurements have shown very high values (millimolar range) which are attributed to impurities (DD Lasic, unpublished data). Normally, covalently
liposome bilayers contain 5mol% lipid with attached PEG with a molecular mass of 2000 Da
With the development of monoclonal antibodies and long-circulating liposomes, the targeting of their contents to particular cells become feasible. In addition to classical techniques used to link antibodies, lectins or other ligands to phosphatidylethanolamine, coupling to the far end of the PEG chain has been developed. The inert terminal methoxy group is replaced with a reactive functional group suitable for conjugation after the liposomes have been prepared containing such a reactive polymer-lipid. Antibodies can be coupled to liposomes in several ways (Fig. 3) [18*].
Some experimental
results and discussion
The hypothesis of increased repulsive pressure above membrane and reduced adsorption on the blood circulation lifetimes was tested by measuring the repulsive pressure between membranes with and without incorporated polymer-bearing lipid by using the osmotic stress technique. Figure 4 shows that bilayers containing PEG lipid show much larger interbilayer spacings, and even upon strong compression the bilayers are still spaced 4nm apart compared with surface unmodified bilayers, which show practical collapse to the hard wall (Born) repulsion [ 19,201. The interbilayer repulsion can be calculated from the repulsive pressure of surface-attached polymer in a mushroom configuration according to:
P - (5/2) kTNlDz/a (a/(A/2))w3 where N-44, size of the monomer a -0.35 nm, and distance between grafting points D==3.57nm; k is Boltzmann constant and T is temperature. The value obtained is in good agreement with experimental data [19]. For distances A>& (where A, is the thickness of the polymer layer), the repulsive pressure is zero. Theoretical extension of this simple law leads to the parabolic decay instead of steep single-step decay at /I>&. The sensitivity of these results, however, cannot distinguish between
394
Biomaterials
Figure 2
0
mPEG-0 +
N-PE H
0
I
%-PEG
mPEGdichlorotxiazine mPEG
Rl
/d
K-PEG p+
mPEG-0
S:fPEG
N-PE H
PE
mPEG-tresylate i rnPEG,
,PE z
1
Several different reaction, pathways for the conjugation of PEG to diacyl phosphatidylethanolamines.
The linkage obtained
by tresylate reaction
(PEG-NH-), as shown on the bottom and cited in all available literature, will have to be revised in the light of a recent finding [56] relatmg to the PEG-O-OSO-HCH-CO-NHbond (S Zalipsky, personal commumcation), which is considerably less stable than the originally proposed secondary
amine linkage. (Figure courtesy of S Zalipsky.)
parabolic or step decay, indicating that the simple scaling approximation is rather good [ZO]. Similar data (Fig. 4) have also been obtained using the surface force apparatus [ZO]. These results also show increased repulsion with increasing amount of PEG polymer. Surface force measurements have found reversible repulsive force at all separations, and the thickness of the steric barrier was found to be controlled by the amount of added PEG-lipid [ZO].
where RR is the radius of gyration of the polymer in a theta solvent and corresponds to the thickness of extending polymer and can be substituted by Rb. (Flory radius=nf@lj) in good solvents. Despite an underestimation of the polymer layer thickness in the low coverage regimen, both models are able to fit the force-distance profiles rather well (201, as shown on Figure 5. Scaling analysis fit, which also describes the measured dependence rather satisfactorily, can be expressed as: F,(//)/R = 1.6 (2 kT/Dz) [(R,:/h)s/J -11
At low coverages (dilute mushroom), Dolan and Edwards mean-field theory of steric forces and scaling model described experimental data satisfactorily. At higher coverages, Alexander-deGennes theory based on scaling concepts described the data better, and a more complex hlilner-Witten-Cates mean-field treatment [21] did not improve the fit (201. At low coverages and in the mushroom model, the Dolan Edwards mean field expression for force between two curved cylindrical surfaces of radius K can be described by:
At higher grafting densities, the force could be described
FJh)/R = 72 (kTID2) exp (--h/R,)
n, = D (R,:lD).Y~
where the numerical
prefactor
is close to expected
unity
[201. that is, in the brush regime, by:
F,(h)/R = (16 kTn /I,)/ (3503) [ 7(2/i,/b)~~~ + 5 (h/2/1,)7/4 -121 where
Liposomes and biopolymers in dnrg and Qene deliiwn
lasti and Papahadjopoulos
395
Figure 3
Biotia-DOPE AbBiotin
+
Avidin
+
A&SH
Ab-Biotin-Avidin-Biotia-Liporomer
p
Biotinyhtcd@somcs
Biotinyhtedantibody
MPB-DOPE
Biotio-Liporomcs
Immlmoli~
0
+
PDP-DOPE
::
R
S-S-fCI-&-C-NH-DOPE-Liposomc
S-(CH,h-C-NH-DOPbLiposomer MPB-antibody
PDP-PE Ii-
Hz-PEG-DSPE
Ab-CHO
+
f:
NH~NH-C-CH2-NH-C-O-[(CH~~-O]~-C-NH-DSPE-Liposomea
Oxidizedantibody
Hydmzidc-PEG-PEliposunes 1 :: :: :: Ab-CH+4NH--C--CH2-NH-C-O-[(CH2~-O]a-C-NH-DSPE-Liposomes Imfnd~
PDP-PEG-DSPE
::
%
S-S--fCH,),-C-NH--_I(CH2~O].-C-NH-DSPE-Liposomes
MPB-8lltibOdy
PDP-PEGPE liposomcs
P
::
S-(CHzh-C-NH-_[(CH&O].-C-NH-DSPE-Lipoaomcr
Several different reaction schemes for the attachment of antibodiis onto liposomes. Abbreviation are as follows: Ab is antibody, DOPE is dioleoyl phoaphatidylethanolamine, MPB is (maleimidophenyl)butyroyl, PDP is (pyridylithio)propionoyl, Hz is hydrazide, PEG is poly(ethylene) glycol and PE is phosphatidylethanolamine. (Published with permission from 1161.)
The force between two cylindrical surfaces (F,) and repulsive pressure (P), as measured by the osmotic
stress technique, approximation:
can be calculated
using the Derjaguin
Liposomes and biopolymen
Figure 5
in drug and gene dolivery Lasic and Papahadjopoulos
397
The first therapeutic studies were performed with epirubicinand doxorubicin-containing liposomes. The results obtained were very encouraging: whereas treatment with free drug and drug encapsulated in conventional liposomes did not reduce tumor growth, treatment with the drug administered in sterically stabilized liposomes caused complete remission of the implanted tumors (281. These studies, which were performed in mice models of colon carcinoma and mammary tumors, yielded complete remission of slowly growing tumors and of the cytostatic effect of quickly growing tumors, and were followed by reports of effective suppression of the growth of human xenografts in nude mice [SO]. These data have been reviewed [29-331.
The explanation for this effect is simple: tumor tissue is often characterized by badly formed and leaky vasculature. Small particles, which can circulate for prolonged periods of time, therefore can accumulate in sites where the blood vessels allow extravasation. Indeed, studies of tumor accumulation of drug encapsulated in sterically stabilized liposomes have shown that up to 10% of an injected dose appears in the neoplastic tissue after systemic administration. Obviously, this passive targeting concept can be used in the treatment of other diseases, such as inflammations and infections in which the body itself increases vascular permeability to allow influx of repair cells and efflux of damaged material [34,35].
I
I
I
I
I
Successful pre-clinical efficacy studies have been reproduced in humans, and before the commercial availability in late 1995, more than 1500 patients with Kaposi sarcoma were treated with doxorubicin encapsulated in sterically stabilized liposomes. This preparation eventually became the first liposomal therapeutic to be approved by the USA FDA (Federal Drug Administration) in November 1995. The formulation is now being tested in several other tumor models.
8
9.0% =PEG-DSPE
_A-dG
Although we have concentrated mostly on liposome stabilization, surface-decorated liposomes can serve a variety of other purposes. The conjugation of antibodies, lectins, oligosaccharides and other (macro)molecules afford liposomes specific reactivity with specific molecules, polymers, surfaces or cells and appropriate receptors on their surface [3].
11
g,o_/ , ,MwcJyo;,o ,. 1 6
8
10
12
14
16
18
Distance, D (nm)
Theoretical fit of force-distance profiles obtained by surface force apparatus. The Dolan and Edwards (D&E), deGennes (dG) and Milner-Witten-Cates (MWC) models are used. (Adapted from [201.)
Earlier attempts at antibody-directed targeting of liposomes [36-391 had severe limitations with regard to applications in who because of their rapid non-specific uptake by the cells of the reticuloendothelial system. It was therefore important to establish whether steric stabilization could be used in conjunction with surface ligands such as antibodies to achieve cell-specific targeting in vivo.
398
Biomaterials
An early observation with sterically stabilized liposomes was that their accumulation in implanted mouse tumors was further increased when tumor-recognizing antibodies were conjugated to the liposome surface [40]. The ability to target these liposomes to other specific tissues by specific ligands was clearly demonstrated using antibody-recognizing lung endothelial cells [41]. Recent studies from several laboratories have shown that sterically stabilized liposomes bearing antibodies to specific cells do not lose the ability of long circulation times in blood and, additionally, anti-tumor activity, if injected at an early stage in tumor development [42,43]. This is quite encouraging with regard to the future possibilities of targeting in vtio, and is of obvious significance in tissues where these liposomes can penetrate to a certain extent the target cells, such as liver, spleen, areas of inflammation and possibly in some tumors. Immunogenicity of these liposomes (i.e. their clearance from blood after repeated administrations), however, has not been studied yet.
Although sterically stabilized liposomes are known to localize within implanted tumors beyond the endothelial layer [44,45], it is not possible to predict whether liganddirected targeting will greatly enhance their accumulation and/or their efficacy in solid tumors. This is because solid tumors do not have an efficient drainage system and allow only limited diffusion of large macromolecular assemtjlies such as liposomes [45,46]. Any extravasated particles, therefore, are not expected to have access to the majority of tumor cells for directed delivery of their contents. In this context, the release of small diffusible drug molecules from the liposome interior would be important for the delivery of cytotoxic material to other proximal tumor cells in the surrounding area. Some approaches to programmable release of the encapsulated agents, which rely on direct or indirect polymer-liposome interaction, are described below. Data from therapeutic experiments demonstrating increased antitumor efficacy against mouse tumors not sensitive to the free drug have confirmed this hypothesis and have provided the basis for clinical trials [28,43,47-SO].
In addition to stabilization and targeting, surface active groups can induce vesicle aggregation, and fusion among themselves or with cells, and can control leakage and possibly phase behavior. Inclusion of lipids with timedependent conjugated charge or polymer can generate additional ways of regulating colloidal and mechanical liposome stability and phase behavior. For example, bilayers composed of micelle- and inverse-micelle-forming molecules, such as PEG-lipids and dioleoyl phosphatidylethanolamine, form stable bilayers. However, if the PEG polymer is cleaved-by hydrolysis of a boad with known reaction kinetics, by photo-, heat-,
or pH-induced trigger, or by simply the dissociation of the whole molecule from the bilayer because of shorter acyl chain(s), for example-phase transition occurs and encapsulated material is released (S Zalipsky, D Papahadjopoulos and collaborators, unpublished data). In addition to lamellar-hexagonal phase transition, one can induce lamellar-micellar transition by cleaving acyl chains of the lipids. Fusion or liposome dissolution (lamellar-micellar phase transition) can be induced also by free polymers which change their conformation as a function of pH [51,X].
PEG-lipids titrated into lipid bilayers have also shown some other applications, such as in the formation of stable emulsions [53-551. The observation that such bilayers can generate extremely stable foam [29] has not been investigated further. Two other cases of liposome interactions with non-conjugated polymers can be mentioned: the ability of free PEG polymers to induce liposome fusion; and polyelectrolyte-oppositively charged liposome interaction.
The most rapidly developing field in the applications of liposomes and other colloidal and macromolecular particles is their role in the delivery of DNA plasmids or short single-strand oligonucleotides with antisense sequences into cells. Mostly cationic liposomes and some positively charged polyelectrolytes, or their combinations, are used to condense and deliver DNA into cells. The structures of cationic lipid-DNA complexes are not yet understood. Because of unwanted toxic effects and lack of specificity and stability, we believe that cationic liposome-DNA complexes are only an intermediate solution. and in the long run these complexes will be coated with anionic lipids and possibly surface-grafted polymers as well as targeting ligands. Some other polycations, cationic polypeptides or polyelectrolytes will find use in DNA condensation and enhancement of encapsulation in anionic liposomes as well as some macromolecules to interfere with endosomal disintegration of endocytosed material or induce direct fusion. Therefore, in this area, surface modification and biocompatible polymers will represent a major development of such ‘artificial viruses’. It is hoped that the interplay of colloid and polymer science coupled with organic synthesis will lead to rational design of ‘intelligent’ colloidal systems and novel applications.
Undoubtedly, phase, colloidal, and biological stability and interaction characteristics of liposomes can be further improved and tailored by coupling appropriate macromolecules, possibly with time-dependent membrane association kinetics, onto their surfaces. We believe that such improvements will lead to novel drug and gene delivery systems.
Liposomes end biopolymers in drug and gene delivery Lasic and Papahadjopoulos
References
and recommended
reading
23.
l.
Kenworthy AK, Simon SA, Macintosh TJ: Structura and phase
behavior of lipid suspensions contalnlng phospholipids with covalently attached PEG Biophys I 1 QQ5,68:1903-1920.
Papars of particular interest, published within the annual period of review, have been highlighted as: .
399
24.
Kenworthy AK, Hristova K, Needhsm D, Macintosh TJ: Range and magnitude of sterlc sterlc pressure between bilayers containing phospholiplds with covakntly atfached PEG Biophys J 1995, 68:1920.
of special interest of outstanding interest
1.
Banghem AD, Hill MW, Miller NGA: Preparation and use of liposomes as models of biological membranes. Methods Membr BiollQ73, 1 :I -68.
25.
Lssic DD, WoodIe MC, Martin FJ, Valentincic T: Phase diagram study of ‘Staalth’-lipid tactthin mixtures. Period Bill 1981, 27:28?-281.
2.
Papehadjopoutos D (Ed): Liposornes and their use in biology. Ann NY Acsd Sci 1870, 378:1-412.
26.
Woodte MC, Cotlins LR, Sponsler E, Kossovski N, Pspahadjopoulos D, Martin FJ: Sfarlully stabtltzad liposomes:
3.
Lasic DD: Liposomes: from Physics to Applications. Amsterdam: Elsevier; 1QQ3:1-575.
4.
Papshadjopoulos D, Allen TM, Gsbizon A, Mayhew E, Hu~I~~Q SK. Woodle MC. Lssii DD. Redemenn C. Martin FJ: Stertcallv stablIlted Ilpo&mes: improvements in ‘pharmacokinetics &d antttumor tharapautk efficacy. Proc Nat/ Acad Sci USA 1991, 88:11460-l 1464.
Lesic DD, Martin F (Eds): Stealth Liposomes. CRC Press: Bocs 5. . Raton; 1 QQ5:1-289. Reviews the physics, chemistry, biology, phannscology and rnedii applications of stericslly stabitiied liposomes. 6.
Felgner PL, Gadek TR, Holm M, Roman R, Wenz M, Northrop J, Ringold M, Dsnielsen H: Lipofactton: a highly effictent lipidmediated DNA transfectton process. Proc /Vat/ Acad Sci USA 198?,84:?413-7417.
7
Lssic DD, Bsrenholz Y (Eds): Liposomes: From Gene Delivery and Diagnostics to Ecology. Boca Raton: CRC Press; 1 QQ6:1-389.
8.
Minsky A, Ghirlando R, Gems& H: Structural features of DNA-c&tonic liposome complexes. In Liposomes: from Gene Delivery and Diagnostics to Ecology. Edited by Lssic DD, Berenholz Y. Bocs Raton: CRC Press; 19QS:?-30.
Q.
Gao X, Huang L: Potentration of &tonic mediated gene delivery by polycattons. 35:1027-l 036.
liposomeBiochemistry 1996,
Lssic DD, Gabiion A, Huang K, Martin FJ, Pspshad$poulos
D:
Sterically stabilized liposomes: a hypothesis on the molecular origin of extended circulation timas. Biochim Biophys Acta IQQI,
1070:18?-192.
28.
Husng SK, Mayhew E, Giteni S, Lasic DD, Martin FJ, Pspshadjopoub D: Pharmacokinattcs and thampautks of sterkalfy stabiltzed liposomas In mtca with C-26 colon carcinoma Center Res 1982, 52:6??4-6781.
29.
Lssic DD: Starkally stabillzad 1994,33:1685-l 699.
30.
Paoehsdiilos D: Stealth lloosomes: from stark stablltxatlon to ‘farg&g. In Liposomes: F&m Gene Delivery and Diagnostics to Ecology. Edited by Lasic DD, Barenholz Y. Boca Rston: CRC Press; 1 Q96:1-6.
31.
Woodle MC, Lssic DD: Stertcally stabillzad Siophys Acta 19Q2, 1113:1?1-1 QQ.
32.
Working P, Newman M, HU~~IQ SK, Vsage J, Mayhew E, LaGi DD:
vesiclas
Angew Chem lnt Ed Eng
liposomes.
Biochim
Pharmacokinetlcs, biodlstribution and therapeutic efficacy of doxorubicin encapsulated in stealth liposomas. J Liposome Res 1994, 41667-682.
34.
Dewhirst D. Needhem D: Gttravasatton of steatth Iloosomes into tumom. In Liposomes: From Gene Delivery and~l)&nosfics to Ecology. Edited by Lasic DD, Bsrenhotz Y. Bocs Raton: CRC Press; 1 QQ6:12?-138.
35.
Huano SK. Martin FJ. Friend DS. Psoshsdiitos D: Mechanism of stialth ‘llposome’rccumulatkr~ in so& pathologkal ttssues. In Liposomes: From Gene Delivery and Diagnostics to fco/ogy. Edited by Lssic DD, Bsrenhob Y. Bocs Raton: CRC Press; 1996:ll Q-l 26.
36.
Heath TD, Fraley R, Papshadjopoulos D: Antibody targetfng of liposomes: call spectftclty obfalned by conjugatton of (Fab’), to vesicle surface. Science 1980, 210:539-541.
37.
Silvius JR, Zuckermann MJ: lnterbilayer transfer of phospholipid anchored macromolecules via monomer diffusion. Biochemistry 1993, 32:3153-3159.
Wolff B. Greooriadis G: The use of monoclonal anttbodv Thy1 gdl for”the targeting of liposomas to AKR-A cells in vftro and in vfvo. Biochim Siophys Acta 1984, 802:259-266.
38.
Hansen EC, Kso GY, Masse E, Allen TM, Zstipsky S: Attachment of anttbodtes to sterkally stabilized liposomes: evaluatton, comparison and optimization of coupling procedures. B&him Biophys Acta 1995, 1239:133-l 44.
Leserman LD, Barbet J. Kouritsky FM, Weinstein JN: Targeting to cells of fluorescent llposomes covalengy coupled with monoclonal antibody of protein A. Nature 1980, 288602-604.
39.
Huang A, Huang L, Kennel SJ: Monoclonal antibody covalently coupled with fatty acid. J Biol Chem 1980, 255:8015-8020.
40.
Pspshadjopoulos D, Gab&n A: Targeting of liposomes cells in vfvo. Ann NY Acad Sci 1987, 507:64-71.
41.
Maruysma K. Kennel, S, Huang L: Lipid composttion is important for highly effident target binding and retention of immunoliposomes. Proc Nat/ Acad Sci USA 1990, 8715744-5747.
42.
Ahmad I, Longenecker M, Samuel J, Allen TM: Antibody targeted delivery of doxorublcln in stealth liposomes can eradicate lung cancer in mice. Cancer Res 1993, 55:1484-l 487
43.
Park JW, Hong K, Carter P, Asgari H, Guo LY, Keller GA, Wirth C, Shstaby R, Knotts C, Wood WI, Pspshadjopoulos D: Development of anti pl85Hgss immunoliposomes for cancer therapy. Proc Nat/ Acad Sci USA 1995, 92:1327-l 331.
44.
Huang SK, Lee K, Hong K, Friend DS, Papshadjopoulos D: Microscopic localixation of sterkally stabilized llposomes in colon carcinoma-bearing mice. Cancer Res 1992, 52:5135-5139.
11.
De Gennes PG: Polymers at intarface. Sci 1987, 27:18Q-209.
12.
Zalipsky S: Polyethylene glycol-lipid conjugates. In Liposomes: From Gene Delivery and Diagnostics to Ecology. Edited by Lssic DD, Berenholz Y. Boca Raton: CRC Press; 1 QQ6:Q3-102.
13.
Woodle MC, Engben CM, Zslipsky S: New amphiphatk polymer-lipid conjugates forming long-clrculattng RBS evadlng liposomas. Bioconjug Chem 1 Q94, 5:493-496.
14.
Torchilin VP, Shtilmsn MI, Trubetskoy VS, Whitemsn K, Milstein AM: Amphiphilic vinyl polymars effactively prolong liposome circulation time in vfvo. Biochim Biophys Acta 1994, 1195:181-184.
17
27
Atlen TM: Long-circulating starkally stabllizad liposomes for targeted drug delivery. Trends Pharma Sci 1994, 15:215-220.
Napper DH: Polymer Stabilization of Colloidal Dispersions. New York: Academic Press; 1983.
16.
of electrophorettc moblllty but not surfaca potential.
Biophys J 1 QQ2,61:902-907.
33.
IO.
15.
re&cU&
Adv Colloid interface
PS Uster, TM Allen, BE Daniel, CJ Mender, MS Newman, GZ Zhu: FEBS Lett 1996, in press.
18. Lssic DD, Needhsrn D: Stealth ltposomes - a prototypical . biomaterial. Chem Rev 1995, 95:1601-l 628. A review of the mechanical and sterical properties of liposomes. 19.
Needhsm D, Hristova K, Macintosh TJ, Dewhirst D, Wu N, Lssic DD: Polymer grafted liposomes: physical basis for the stealth property. J Liposome Res 1992, 2:41 l-439.
20.
Kuhl T. Leckbsnd D . L&c DD. lsraelechvili JN: Modulatton of Interaction forces b&en bilayers exposing short chained ethylene oxide head groups. Biophys J 1984, 66:1479-l 488.
21.
Milner S: Polymer brushes.
22.
Baekrnark TR, Elsnder G, Lssic DD, Sackmann E: Conformational transitions In monolayers of phospholipld-polyethyteneoxtde lipo-polymers and interaction forces wtth solid surfaces. Langmuir 1995, 11:39?5-3987.
Science
1991, 251:205-210.
to tumor
400
Biomateriais
45.
Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D. Jain RK: Microvascular permeability and interstitial penetration of stericaiiy stabilized iiposomes in a human tumor xenograph. Cancer Res 1994, 54:3352-3359.
51.
Thomas U, Tirrell DA: Poiyeiectroiyta sensitized vesicles. Act Chem Res 1992, 25~336-342.
52.
46.
Wu NZ, Da D, Rudolf TL, Naadham D, Dewhirst M: increased microvascular permeability contributes to preferential accumulation of stealth ilposomes in tumor tissue. Cancer Res 1993,53:3?65-3770.
Parente RA, Nir S, Szoka FC: Mechanism of leakage of phosphoiipid vesicles induced by the peptide GALA Biochemistry 1990, 29:1720-l 728.
53.
47.
Mayhew E, Lasic DD, Babbar S, Martin FJ: Pharmacokinetics and antitumor activity of epirubicin encapsulated in iongcirculating iiposomes. Int J Cancer 1992, 51:302-309.
Wheeler JJ, Wong K, Ansall SM, Masin D, Baily MB: Polyethylene giycoi modified phosphoiipids stabilize emulsions prepared from triacyigiyceroi. J Pharm Sci 1994, 83:1558-l 564.
54.
48.
Vaage J, Mayhew E, Lasic DD, Martin FJ: Therapy of primary and metastatic mouse mammary carcinoma by doxorublcin encapsulated in stericaiiy stabilized iiposomes. Int J Cancer 1992, 51~942-947.
La&c DD: Afterword and conclusion. In Stealth Lioosomes. Edited by Lasic DD, Martin F. Boca Raton: CRC Press; 1995:279-280.
55.
Warriner HE, idziak SH, Slack NL, Davidson P, Safinya CR: Lameiiar biogeis: fluid membrane based hydrogeis containing polymer lipids. Science 1996, 271:969-973.
56.
Gais HJ, Ruppart S: Modification and immobilization of protelns with PEG tresyiate and poiysaccharide tresyiates: evidence suggesting a revision of the coupling mechanism end the structure of the polymer-polymer linkage. Tetrahedron Lett 1995,36:2837-2838
49.
50.
Williams SS, Aiosco TR, Mayhew E, Lasic DD, Martin FJ, Banked RB: Arrest of human lung tumor xenograft growth in SCID mice using doxorubicln encapsulated in stericaliy stabilized iiposomes. Cancer Res 1993,53:3954-3958. Allen TM, Mehra T, Hansen C, Chin YC: Stealth liposomes: an improved sustained reiese system for arabinofuranosyicytosine. Cancer Res 1992, 52:2431-2436.
phosphoilpid