METHOD FOR THE PREPARATION OF MICROSPHERES WHICH CONTAIN COLLOIDAL SYSTEMS

Abstract: The invention relates to a method for the preparation of microencapsulated colloidal systems such as liposomes, <i>i.e.</i>, microspheres which comprise colloidal systems. These microencapsulated colloidal systems can be used as controlled release systems for the delivery of active ingredients in <i>in vivo</i> and <i>in vitro</i> applications. A method is provided in which the colloidal systems are added to a phase which comprises a water soluble crosslinkable polymer followed by formation of microspheres.

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Φ X B rH J XI > SH 4-J CQ β CJ cn X T) 4-J 4-J ( 0 ε SH φ ft ti 4-J CQ X! 4-J φ O 3 Φ Cn (0 O O -H -H -rl 0 - CQ Φ Φ • H rd Pi cn r 0 X! 4-J X! 4-J Φ H CQ ε ■U O SH rt V CQ β 4H β β TS rH CD -H T ) xl 4J A ε β rd • CQ 4-1 rH β ε Φ rO CJ φ β -H T5 SH -rl CQ O φ 0 • H U SH CQ o -rl -H 4-J ti 3 3 td 0 4-> 4H Φ rd r- -rl rl ε -H O CQ 4H ε -H -rl β ti ε 4H -rl rd Tl ε > SH ft 0 O r-i 4-J u u 05 CQ O x; CQ ε 0 rl (0 β Φ 4H Φ 4-J rH A ti Φ 0 ti ft Φ Φ 4H (0 Φ SH -rl Φ r-i 03 - l Pi 4-J 4-J ε TI O CQ r-i (0 4 -rl ft u rO 4-J β 4-J υ XI 4-J 0 > CJ -H XI tn β ro CQ Φ φ (0 β 4H - υ CQ 4-1 ft φ ffi CQ β Φ Φ CQ 03 CQ td CQ (0 rd 4-J 0 -H o β β rH 3s tO CD Φ -H 4H Pi SH 4- ) Pi 0 SH Pi rH β CQ B XI X! β 1 r-i ro B o -H ft . o Φ β CJ 4-J O CQ os o • CQ -H O rd CQ ft -rl φ 05 Φ -H r TI o TI O O TI r-i CQ -i -rl β ti U β -H 2 4-J q φ φ SH CQ XI φ r-i φ J Φ ro ti • CQ ti Φ Φ J φ rO ( 0 Φ Φ •rl PQ Φ 3 U CO β SH 4J O 3 Φ 4-J rd 4J td CQ H 4H TS 4-J J CQ «. β • rl XI 4-J ft rH CQ Pi r3 CQ rH β 35 0 ft O ε β 0 β 0 ε 3 CN φ Φ SH rl -H 4-J CQ ft rd 4-J CQ » rO O < •rl o CQ SH Φ 0 0 SH o β ^ CQ XI r-i φ Φ φ CJ ^ CQ XI SH -H X! -rl CJ ^ • X! CQ φ β • H ft 4H J CQ -rl ft ■ rl 03 Φ o Φ -rl ft x: ft X! ϋ •rl 3 Φ J ft β -rl ti TS ft • 0-1 X 0 ( 0 4J 4-> CQ x; XI 1 r-i ti 0 4-J CQ 4-J rO ft CO I β 1 -H ft 4J ω 1 CQ TS O SH Φ β «. ft Φ -H • H ft CJ CJ rl -rl SH > cn • H O 4H 0 CQ O 3 φ ti <ϋ Φ A CQ -rl CQ SH XI TJ 0 1 -rl CQ τ) «. T) 3 Φ β TS r-i ^H Si 4H H • H » s O X U CQ i, 4-J 4-J φ Φ 0 4-J TS 4-J (0 4-J U 4-> φ Pi fc XI -H φ O ft 4J ; ε O 4-J Φ - l Φ β 4-J J CQ rd -rl W SH TS 4H r-i φ CQ X > ft • • Φ β 3 ε . X! rH 4H 3 0 rO XI 4H ε φ rd 0 4-J XI • 4-J 4-J r-i Φ tn .. β X! 0 rO σ Ti EH 0 0 O >H 4H rO 0 O - 0 r- β ft CQ β Ti β 0 O 0 r- X! β β CJ -H rO Φ φ • ft ε ft SH H rl SH A -H ε φ O φ Φ 4 £ CQ (0 EH -H 0 4-J * . SH ε . CQ rH 03 3 TS φ 0 4H 03 ti rl 3 4-J > ε CQ u β CQ CQ ,-, rO Φ β rd SH CQ β S rO Φ CQ Φ CQ -H 4-J Φ CQ 0 y rH rO Ti ■ -H -H -H CQ ε rH J r- rO O ε -rl β > φ X! 4-J rO > 1 - l ε Φ 4H Φ Φ O CJ φ Φ Tl ti 03 β Φ 0 • H J ^ -, 4H Φ φ Q -rl © 0 0 cn 4-J TJ - l Φ tO 3 pi, CQ CQ CQ C Tl CQ Φ 4-) ( 0 4-J ϋ rH -H β ϋ 4J ■P ϋ ε 03 ε rH X! φ CQ -i rd φ XI (0 CQ Ti β CO Φ XI β A CQ Pi ft 4J Φ ω Φ CQ 0 φ φ td 4H φ -H φ Φ SH β 0 X! A 3 XI -rl φ 0 X! XI ft O O Pi rH ft SH : U SH Pi rl CQ ti rH O XI r-i ti XI rO 3 ft ft 4-J CO ft Ti ε XI ft 4-J CQ u rD O cn ^ rO • s rd CO ft Liposomes have been under investigation as controlled release systems for many years. Liposomes consist of concentric closed membranes, such as a bilayer formed by water-insoluble polar lipids, such as phospholipids . Other substances, such as cholesterol, may also be included in the membrane. These liposomes may vary in diameter from a few tens of nanometers up to micrometers. Both polar and non- polar drugs or enzymes can be encapsulated as active ingredient within liposomes. Proteins can be encapsulated in liposomes very well. When using liposomes as controlled release systems, initial high concentrations of proteins in the blood may be prevented. Such high concentrations are often undesirable, because they may lead to adverse effects in the patient and to degradation of the protein. The release of the active ingredient from the liposome may be triggered by the destabilization of the bilayer. (Micro) encapsulation of liposomes has been reported in several instances. For example, Kibat et al . (FASEB J. , 4 (1990) 2533-2539) describe microcapsules composed of a hydrogel matrix of calcium alginate . These microcapsules contain liposomes. Pulsatile release of compounds from the liposomes is obtained by coating the liposomes with phospholipase A 2 enzyme. Cohen et al . (Proc. Natl. Acad. Sci. USA, 88 (1991) 10440-10444) and Cohen et al . (Proceed. Intern. Symp . Control. Rel . Bioact . Mater. 16. (1989) 71-72) also describe alginate-microencapsulated liposomes. These microencapsulated liposomes are characterized by a release profile which starts immediately, i.e., show no lag time. In addition, the release is gradual rather than pulsed. US-A-4, 921, 757 discloses liposomes that are encapsulated in a solid permeable plate of e . g. agarose or polyacrylamide, or in microparticles based on crosslinked alginate with a polykationogenic skin. Yeung et al . (J. Microencapsulation, 5 (1988) 331-337) describe liposomes incorporated in nylon based microcapsules, which are prepared using a interfacial polycondensation process. The release profiles of these microencapsulated liposomes shows no lag-time. Moreover, the use of nylon makes in vivo application unlikely. Bochot et al . (International Journal of Pharmaceutics, 162 (1998) 119-127) describe an ocular delivery system based on liposomes dispersed in a thermosensitive polyoxyethylene- polyoxypropylene copolymer gel . The system is prepared by adding liposomes to the gel. Weiner et al . (Journal of Pharmaceutical Sciences, 74 . (1985) 922-925) describe the use of a collagen gel for encapsulating liposomes. After the liposomes have been added to the collagen, gel formation is initiated by changing the temperature or the pH. Alamelu et al . (Carbohydrate Polymers, 24 . (1994) 215- 221) describe a delivery system based on sequestered liposomes coupled to a chitosan gel. Gel formation is effected by addition of an alkaline solution. Finally, DiTizio et al . (Biomaterials, 19 (1998) 1877- 1884) describe a liposomal hydrogel. The gel is formed by changing the pH. All of these prior art microencapsulated liposomes rely on enzymes to effect the release of compounds from the liposomes or do not show desirable release characteristics. In addition, the encapsulation efficiency of the prior art methods is usually too low. The present invention is aimed at providing a new injectable, patient friendly delivery system for active ingredients, such as protein drugs or other drugs, which system is safe and biodegradable, and which system possesses well controllable delivery kinetics. The period wherein drug delivery should be guaranteed depends on the active ingredient used that preferably is protein drug, and varies between a number of days up to more than one year. In addition, high degrees of loading in the delivery system should be obtained. Moreover, the system of the present invention should be produced without needing the use of organic solvents. It is another objective of the present invention to provide a method for the preparation of microencapsulated colloidal systems, such as liposomes, which colloidal systems may be associated with an active .ngredient, which microencapsulated colloidal systems display a desirable release profile, i.e., a high release of active ingredient (preferably more than 85%) , a pulsed release profile having a suitable lag-time, while these release profiles can be controlled by the method of preparation. The desirable release profile should also hold for the active ingredient which may be associated with the colloidal systems. The definition of a suitable lag-time depends on the type of application. When vaccines are used as an active ingredient, a suitable lag-time is between two weeks to one year, preferably 1-3 months. It is another objective of the present invention to provide such a method which enables a high encapsulation efficiency of the colloidal systems, i.e., an encapsulation efficiency of more than 80%, preferably more than 90%. SUMMARY OF THE INVENTION The present invention is directed to a combination of colloidal systems and hydrogels used for controlled release of active ingredients. Suitable colloidal systems which may be used in the present invention are liposomes; iscoms; polyplexes, i.e. combinations of (cationic) polymers and DNA; lipoplexes, i.e. combinations of (cationic) lipids and DNA; nanoparticles, i . e . polymer based spheres in the nanometer size range; solid lipid particles in the colloidal size range (see for example R.H. Muller et al . , Pharm. Research 14 (1997) 458-462); emulsions, such as intralipid-like systems; any other entity in the colloidal size range having a low water solubility; and combinations thereof. The combinations of colloidal systems and hydrogels according to the present invention will be illustrated hereinafter using liposomes as the colloidal system. It is to be understood that were liposomes are mentioned hereinafter, these liposomes can be replaced by any of the suitable colloidal systems mentioned above, without departing from the spirit of the invention. The problems mentioned above are solved by a specific preparation method of controlled release systems, such as microspheres, wherein water is used as the solvent. The use of water as sole solvent system is advantageous from an environmental point of view, because of toxicological considerations and, especially, because of reasons of protein stability. In a first aspect, the present invention provides a method for the preparation of a controlled release system, comprising : (a) forming an aqueous two-phase system from two water soluble polymers and water and at least one releasable entity, such as a liposome or another colloidal system, the two water soluble polymers being incompatible in solution, at least one of these polymers being crosslinkable, the crosslinkable polymer phase being emulsified in the other polymer phase; and the at least one releasable entity being soluble or dispersible in the crosslinkable polymer phase in the aqueous solution; (b) allowing the releasable entity to distribute in the crosslinkable polymer phase; and (c) crosslinking of the crosslinkable polymer. In a preferred embodiment, the crosslinking is carried out to such a degree that the pores (meshes) in the crosslinked structure eventually formed are substantially smaller than the size of the releasable entity. 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For a given starting-composition, the volume ratio of the coexisting phases x x /x 2 equals γ 2 /Y ι - As indicated herein-above, the releasable entity can be a protein drug. However, it is also possible to encapsulate a pharmacon or antigen or other active agents containing colloidal systems, such as nanoparticles or microparticles, e.g. liposomes and iscoms. The encapsulation of this type of particles has the advantage of preventing the occurrence of a too fast release of the encapsulated entity, or, said in other words, burst-effects can be avoided in a more secure way . The microencapsulated colloidal systems obtained by the method of the invention comprise a matrix of a degradable polymer in which the colloidal systems, such as liposomes, are present. Active ingredients may be present in the colloidal systems. It is a considerable advantage of the present invention that these active ingredients may be chosen in principle regardless their compatibility with the polymeric matrix, i.e. it is not required that the active ingredients are soluble or dispersible in the matrix itself, since it is sufficient that these active ingredients are compatible with the colloidal system. This enables the preparation of release systems comprising active ingredients which could only with difficulty or only with additional measures, be incorporated in release systems of the prior art . The method of the invention thus enables microspheres for e . g. the delayed or pulsed release of all types of active ingredients. When liposomes are used as the colloidal systems, the presence of the bimolecular lipid membrane layer enables the incorporation of hydrophobic substances, which could not be brought in conventional hydrogel systems. These hydrophobic substances can be incorporated in, or associated with the lipid bilayer. Examples of colloidal particles to be encapsulated are: iscoms, lipoplexes, polyplexes, nanoparticles and solid liponanoparticles . Iscoms may be released by the microspheres slowly or in a pulsed manner. This allows the designer of vaccines to manipulate the immune response induced by the antigen- carrying iscoms. E. g. , if a booster is desired, the microspheres can induce a booster effect by pulsed release of the iscoms after a predetermined delay time. It may be desired to have access to delivery systems that release polyplexes and lipoplexes (complexes of genetic material and condensing polymer (mixtures) or (mixtures) in a time controlled way. The microspheres are able to do that. The same applies to nanoparticles and lipospheres, which can be loaded with therapeutically active material or antigens. Nanoparticles are colloidal particles based on e . g. polycyanoacrylates . Solid lipid nanoparticles are colloidal particles based on lipids which are in the solid phase at body temperature. Nanoparticles and solid lipid nanoparticles can be released from the microspheres in similar patterns as liposomes. Drug release from these colloidal particles depends on their building components and manufacturing conditions. Moreover, by incorporating the active ingredient in the colloidal system, the active ingredient is protected from the reactions that take place during the subsequent polymerization step, which reactions could cause undesired oxidation of the active ingredient. With the method of the invention, it is possible to provide microcapsules which comprise 10 wt . % or more of the colloidal system. Usually, the amount of incorporated colloidal system will be from about 5-10 wt . % but this amount will vary with the specific conditions used. 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SH •rl ( 0 SH Φ CQ β A CQ cn rd CO ti ft • H rH CQ Φ β CQ 0 β Φ X! 0 φ cn 4H T) 0 Φ N N Φ X td 0 Φ β J •H -H 4H -H 0 Xi Φ 3 H ti 0 l iH r-i Φ u β H Φ 4H TJ > •rl •H X H Φ CJ Tl SH -H CQ Φ Φ -i 4H X ft ft -rl CJ -H ft -H 3 β -H -U X! φ TJ -i SH ft rH φ 3 4-S Φ ε XI Φ α -H CQ Tl Φ • H 4-J CQ SH CJ 1 Φ 4 β 4-> Ti N ro fO Φ 0 Φ t J X! ε Pi 4-1 13 i Φ 0 r-i 0 φ X! β 0 rd rd 3 CQ φ r-i -H ε I CJ rl β J CJ Ti 3 4-J 05 r CQ X SH ti Φ ti ε r- 3 CQ 4J 3 i 4J -rl 0 ( 0 Φ 3 TS β r-i Φ cn TJ Φ CJ n β Φ > J -H Φ 4H -r~ι Φ rd 0 •rl fd β r 4-1 φ 4H SH φ 0 4H • H X! XI β (0 X β •rl SH X β Ti SH XI xi X XI -H 0 Xl β 4H 4-1 X! SH CQ -rl Ξ5 H X X 4-J B -H CJ 4-J -rl ε ft 4-J -H fO ft 4J CQ 0 4-J ε ft a 4-1 •H Φ CO EH ft than 2 years. A very preferred drying method is spray-drying, although the drying can also be suitably carried out using lyophilization. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a volume particle size distribution of dextran microspheres prepared via the water-in-water emulsion technique . Fig. 2 shows a SEM micrograph of the dextran microspheres of Fig. 1. Fig. 3 shows the volume weight average diameter of dextran microspheres as a function of the degree of MA substitution and the molecular weight of PEG. Fig. 4 shows the volume weight average diameter of dextran microspheres as a function of the degree of MA substitution and concentration of the aqueous dex-MA concentration. Fig. 5 shows the effect of the concentration and molecular weight of PEG on the volume weight average diameter of dextran microspheres. Fig. 6 shows release profiles of degrading dextran microspheres . Fig. 7 also shows release profiles of degrading dextran microspheres. Fig. 8 shows a phase diagram of a water/PEG/dextran ternary system. Fig. 9 shows liposome release for 3 individually prepared batches of dextran microspheres at pH 7.2. The formulation of the microencapsulated liposomes being: DexHEMA DS 8, water content 70%, PEG/dex volume ratio: 40, 184 nm DPPC:DPPG:Chol (10:1:10) liposomes. Fig. 10 shows the release of free calcein, total calcein, liposomal phosphate at pH 8.0. The formulation at the microencapsulated liposomes being: DexHEMA DS 8, water content: 70%, PEG/dex volume ratio: 40, DPPC :DPPG:Chol (10:1:10) liposomes with (226nm) and without (184 nm) calcein. Fig. 11A shows the effect of liposome size on the release characteristics at pH 7.2. The formulation of the microencapsulated liposomes being : DexHEMA DS 8, water content: 70%, PEG/dex volume ratio: 40, 95 and 184 DPPC :DPPG: Choi (10:1:10) liposomes. Fig. 11B, shows the effect of liposome size on the release characteristics at pH 8.0. The formulation of the microencapsulated liposomes being: DexHEMA DS 8, water content: 70% PEG/dex volume ratio: 40, 95 and 184 DPPC :DPPG: Choi (10:1:10) liposomes. Fig. 12 shows the effect of the PEG/dex volume ratio on the release characteristics at pH 7.2. The formulation of the microencapsulated liposomes being: DexHEMA DS 8, water content: 70% PEG/dex volume ratio: 20, 40, and 80, 184nm DPPC :DPPG: Choi (10:1:10) liposomes. Fig. 13A shows the effect of the water content on the release characteristics of microencapsulated liposomes according to the invention at pH 7.2, compared with release from non-degradable dexMA microspheres. The formulation of the microencapsulated liposomes being: DexHEMA DS 8, water content 70 and 50%, PEG/dex volume ratio: 40, 184 nm DPPC :DPPG: Choi (10:1:10) liposomes. Fig. 13B shows the effect of the water content on the release characteristics at pH 7.2. The formulation microencapsulated liposomes being: DexHEMA DS 5, water content: 70 and 50%, PEG/dex volume ratio: 40, 184 nm DPPC :DPPG: Choi (10:1:10) liposomes. Fig. 14A shows the effect of the degree of substitution on the release characteristics at pH 7.2. The formulation of the microencapsulated liposomes being: DexHEMA DS 5 and 8, water content: 70%, volume ratio: 40, liposome size: 184 nm. Fig. 14B shows the effect of the degree of substitution on the release characteristics at pH 8.0. The formulation of the microencapsulated liposomes being: DexHEMA DS 5, 8 and 16, water content: 70% volume ratio: 40, liposome size: 184 nm. Fig. 15 shows the effect of dextran derivative on the release characteristics at pH 7.2. The formulation of the microencapsulated liposomes being: DexHEMA (DS 5) , mono- and polydiperse dexLactateHEMA (DS 4), water content: 70%, volume ratio: 40, liposome size 184 nm. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for the encapsulation of colloidal systems, such as liposomes, in crosslinked microspheres. The present inventors surprisingly have found, that when microspheres which comprise colloidal systems are prepared according to a method which comprises the steps of: a) providing an aqueous mixture of i) a first phase comprising a water soluble crosslinkable polymer, ii) a second phase comprising a water soluble polymer which is incompatible in solution with the polymer in said first phase, and iii) colloidal systems to be suspended in said first phase; forming an emulsion of said first phase in said second phase, and b) forming microspheres in the emulsion by crosslinking at least part of said crosslinkable polymer, thus forming said microencapsulated colloidal systems, the problems mentioned herein-above can be overcome. In a preferred embodiment, the method of the invention is carried out by first mixing the colloidal systems with said first phase and subsequently contacting this mixture with said second phase. By forming a premixture by contacting said colloidal systems with said first phase in step a) , and subsequently contacting this premixture with said second phase, the colloidal systems can be encapsulated in the microspheres with very high encapsulation efficiencies. O ε P 4H 0 O SH 4H S 2> 4-J PQ β T) 4-S 3 Φ φ 4-J Φ β fd TS 0 r-i CJ 0 CD ft β SH fd -H Φ SH CQ 4-J 4-J 3 rd X 3: ω Φ O 05 • rd ^ - ^ Φ cn o r-i β ^ Φ -H SH CD •• 3 • O Φ CQ ■ H Xi CQ Xl 4-S 4-5 β 4-S fd 0 β SH -rl 0 o 4-J φ • -H ε ε co TS ro 3 β -i X 0 β O a CJ rd > X 4-J r-i 4-S - 03 03 o\° CJ SH O O -H Φ in 4-J Cn tn β 0 β ■U Φ -i 0 β ε 0 r-i Φ •rl -H 4-J SH CQ β β Φ P " i Φ O & A > CJ X ft Φ Φ SH SH CO Φ Φ Φ rO 4-J Q T) 3 fd rd β 35 Φ 3 - r-i oV> Φ <£> O CO SH rH m pyridine) as a catalyst, essentially as described by Van Dijk-Wolthuis et al . in Macromolecules 2.8, (1995) 6317-6322. Dex-PEG was synthesized as follows. mPEG (monomethoxy- polyethylene glycol, M 5000 g/mmol, 5 g, corresponding with 1 mmol hydroxyl groups) and CDI (carbonyldiimidazole, 162 mg, 1 mmol) were dissolved in 100 ml of anhydrous tetrahydrofuran. The solution was stirred overnight at room temperature, followed by the evaporation of the solvent under reduced pressure. Next, the Cl (carbonylimidazole) activated mPEG was added to a solution of dextran T40 (1.7 g) and DMAP (0.35 g) in 50 ml DMSO. This solution was stirred for one week at room temperature. After neutralization of DMAP with HC1, the solution was extensively dialyzed against water and subsequently freeze dried. The product was characterized by gel permeation chromatography and NMR. The degree of substitution amounted to 4 Dex-lactate-HEMA (DS 3) was synthesized as described in the copending European patent application No. 96201821.4. Polyethylene glycol (PEG, varying molecular weight) was dissolved in 0.22 M KCl to a concentration of 12-40% (w/w) . Dex-MA was dissolved in 0.22 M KCl to a concentration of 10-40% (w/w) . Both solutions were flushed with nitrogen for 10 minutes. Next, 4.75 ml of the PEG solution and 0.25 ml of the dex-MA solution were mixed and vortexed (Winn Vortex- Genie, maximum speed) for 1 minute resulting in a water-in- water emulsion with dextran as the inner phase and PEG as the outer phase. After 10 minutes ( (N,N,N' ,N' -tetramethyl- ethylenediamine, 100 μl , 20% (v/v) in 0.22 M KCl, pH adjusted with concentrated HC1 to 7.2) and KPS (potassium peroxydisulfate, 180 μl , 50 mg/ml in water) were added. The emulsion was incubated for 30 minutes at 37 °C to polymerize the dex-MA. The microspheres were washed twice with water and freeze dried. It was demonstrated using in vi tro cell cultures that cytotoxicity of dex-MA is low, similar to the cytotoxicity of dextran, which compound has been used for years as plasma replacing agent in human beings. The particle size (number weight diameter (=∑nd/∑n) and volume weight diameter (=∑nd 4 /∑nd 3 ) , I.e. Edmundson, Particle-size analysis, H.S. Bean, A.H. Beckett and J.E. Carles (eds) in: Advances in Pharmaceutical Sciences vol.2, Academic Press, London 1967, 95-174) and particle size distribution were determined by a laser light blocking technique (Accusizer tm , model 770, Particle Sizing Systems, Santa Barbara, CA, USA) . The shape and surface characteristics (porosity) of the microspheres were established by scanning electron microscopy (SEM) analysis. Figure 1 gives a representative example of particle size distribution of a dextran microsphere batch prepared via the water-in-water emulsion technique as determined using the Accusizer. SEM analysis showed that the particles are perfectly spherical and non-porous (Figure 2) . Figure 3 shows the volume weight average diameter of dextran microspheres as a function of the degree of MA substitution and the molecular weight of PEG. The concentration of the PEG solution was 24% (w/w) ; the concentration of the dex-MA concentration was 20%. It is shown that the particle size increases with decreasing molecular weight of PEG. At a fixed molecular weight of PEG, the particle size slightly decreases with increasing DS . Figure 4 shows the volume weight average diameter of dextran microspheres as a function of the degree of MA substitution and concentration of the aqueous dex-MA concentration. The mean diameter decreases with decreasing dex-MA concentration. Figure 5 shows the effect of the concentration and molecular weight of PEG on the volume weight average diameter of the dextran microspheres. For this evaluation, dex-MA with a DS of 8 in 0.22 M KCl (20%, w/w) was used. It appears that for a given PEG, the largest particles were obtained at a PEG concentration around 24%. Example 2 Microspheres were prepared from the following formulations using the protocol as given in Example 1 and using the following stock solutions: Stock solutions (% in w/w) in 0.22 M KCl: A. PEG 10.000, 24% B. PEG 20.000, 24% C. Dex-MA (DS 13) 20% D. Dex-lactHEMA (DS 3) 20% E. Dex-lactHEMA (DS 3) 10% F. Dex-PEG 20% Table 1 summarizes the results As can be seen, a suitable emulsifier (block-copolymer of dextran and PEG) gives smaller particles with a smaller dispersity (= weight mean diameter/number mean diameter) . Example 3 The release of a model protein from non-degrading dextran microspheres and degradable microspheres was evaluated. The microspheres were rendered degradable by the incorporation of dextranase in dex-MA microspheres. Dex-MA (DS 8) was dissolved in 10 mM phosphate buffer pH 8.0. To 2 ml of this solution a fixed amount of IgG (Immunoglobuline G, 25.6 mg) and a variable amount of dextranase (Sigma D1508; 0, 0.1 and 1 U (1 U releases 1 μmol reducing oligosaccharides per minute at 37°C and pH 6.0)) dextranase were added. This solution was emulsified in an aqueous solution of PEG (M 10.000, concentration 24% (w/w)) in 0.22 M KCl. Thereafter, TEMED (N,N,N' ,N' -tetramethyl- ethylenediamine, 100 μl, 20% (v/v) in 0.22 M KCl, pH adjusted with concentrated HCl to 7.2) and KPS (potassium peroxydisulfate, 180 μl , 50 mg/ml in water) were added. The microspheres were washed with water and dried under a nitrogen flow. An accurately weighed amount (0.3-0.5 g) of microspheres was suspended in 10 ml phosphate buffer pH 5.5 and the amount of protein released in the buffer was determined using the biorad protein assay (M. Bradford. Anal. Biochem. 72 (1976) 248-254) . Figure 6 shows the release profiles. From this figure it is clear that the release of IgG from dextran microspheres can be modulated by dextranase. Example 4 (Reference Example) The release of a model protein (IgG) from degrading dextran microspheres was evaluated. Degradation was established by co-entrapment of dextranase in the microparticles. Methacrylate derivatized dextran (DS=8; 138 mg) was dissolved in 612 μl buffer (10 mM phosphate, 220 M KCl, pH 8.0) . Thereafter, 250 μl of an aqueous solution of IgG (50 mg/ml) and 250 μl of an aqueous solution of dextranase (variable concentration) were added. IgG and Dextranase were dissolved in the same buffer (10 mM phosphate, 220 mM KCl, pH 8.0) . Next, 500 μl of the dexMA, IgG, Dextanase solution was added to 5 ml of an aqueous solution of PEG (molecular weight 10.000 g/mol, concentration 17.5, 24 or 30% w/w) in the same buffer. These phase separated systems were vortexed for 1 minute, followed by the addition of 180 μl potassium peroxodisulphate (50 mg/ml; dissolved in phosphate buffer) and TEMED (N,N,N' ,N' ' -tetramethylethylenediamine, 20% v/v, pH adjusted to 8.0 with HCl) . Next, the samples were incubated for 30 minutes at 37°C to polymerize the DexMA. The particles were collected by centrifugation and washed with water. The particles were resuspended in buffer (5 mM NH 4 Ac, pH 5.5) and incubated at 37 °C. Periodically, samples were withdrawn and analyzed for their protein content (Biorad assay) . Figure 7 shows the release profiles. It can be seen that in the absence of dextranase the cumulative release was less than 10%, indicating that the hydrodyn mic diameter of protein was larger than the hydrogel mesh size. Further, the release rate increases with increasing amount of dextranase in the particles. An increasing amount of dextranase in the particles resulted in an increasing degradation rate. This means that the release of entrapped proteins form dextran particles can be modulated by the degradation rate of the hydrogel matrix. Degradation can be established by addition of an enzyme (dextranase) or by the introduction of hydrolytically labile spacers (e.g. lactate esters) in the crosslinks . Comparative Example 5 grams of dextran in which the dextran chains are derivated with methacrylate groups, having a Mw of 40,000, was dissolved in 45 ml of water. A second solution was prepared, which solution comprised 7 g of polyethylene glycol having a Mw of 6,000 in 45 ml water. At room temperature, the first solution was added to the second solution while stirring. The result was a one phase system, from which no microspheres could be formed. This example illustrates the need to choose the molecular weights and concentrations of the starting materials in such a way that a two-phase system is obtained. In the following Examples 5-9 the following materials were used. Polymer-related materials PEG 10.000 (M w 12.000; M n 8700) and potassium peroxodisulfate were obtained from Merck (Darmstadt, Germany). Dex 40.000 (M w 38800; M„ 16400) and N,N,N',N'- tetramethylethylenediamine were purchased from Fluka (Buchs, Switzerland) . M w and M n refer to the weight and the number average molecular weight, respectively (determined by GPC) . Methacrylate (MA) derivatized dextran (dexMA) with a degree of substitution (DS: the number of MA groups per 100 dextran glucopyranosyl monomer units) of 10 was prepared as described by Van Dijk-Wolthuis et al . in Macromolecules 28 . (1995), 6317-6322. Hydroxyethyl methacrylate (HEMA) derivatized dextran (dexHEMA) with different DS were prepared as described by Van Dijk-Wolthuis et al . The degrees of substitution as determined by 1 H-HMR were 5, 8 and 16. Monodisperse and polydisperse lactate HEMA derivatized dextran (dexLactateHEMA) were prepared according to Cadee et al. (Polymer 40 (1999) 6877-6881) and Van Dijk-Wolthuis et al. and both had a DS of 4. Liposome-related materials Dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were donated by Lipoid GmbH (Ludwigshafen, Germany) . Cholesterol (Choi) and calcein were obtained from Sigma (Rockford, IL, USA) . Chloroform and methanol (p. a.) were purchased from Merck (Darmstadt, Germany) . Example 5 DPPC:DPPG: Choi (10:1:10) liposomes were prepared by lipid film hydration as described in Crommelin et al . ('Liposomes', In: Kreuter, J. (ed.), Colloidal Drug Delivery Systems. Inc. Marcel Dekker (1994), pp. 73-190), with and without calcein, an aqueous marker used at a concentration of 80 mM as the active ingredient. The incorporation of calcein is described in Gregoriadis (ed.), 'Liposome Technology', Vol. I, II and III, 1st and 2nd edition, CRC Press, Boca Raton, FL (1984, 1993) . 10 mM Hepes (N- [2 -hydroxyethyl] piperazine-N' - [4-butane sultonic acid]) buffer containing 0.8% NaCl, adjusted to pH 7.2 was used as aqueous medium (total lipid concentration 81 mM) . Subsequently the liposomes were extruded through 0.6, 0.2, 0.1 and/or 0.05 μm polycarbonate filters to obtain the desired size. The calcein containing liposomes were washed by ultracentrifugation to remove the unentrapped calcein. The phospholipid content was measured by the phosphate determination according to Rousser et al . ('Two-dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorous analysis of spots', Lipids 5 (1970) 494-496). Mean particle size was determined by dynamic light scattering (DLS) . The size, polydispersity index and phosphate or calcein content of the liposomes prepared are presented in Table 2. Table 2 Liposome size, polydispersity index, phosphate content and calcein content of liposomes used. Size Phosphate Cal :eιn Liposome type (nm) PD content (mM) content (mM) DPPC:DPPG: Choi (10 1:10) 370 0.31 36.4 X 184 0.10 35.1 X 95 0.05 27.3 X DPPC : DPPG: Choi (10 1:10) 783 0.62 nd 36 226 0.18 nd 39 148 0.11 nd 29 nd = not determined Example 6 Microencapsulated liposomes were prepared by mixing liposomes prepared in Example 5 with an aqueous solution of dexMA in 10 mM Hepes buffer. Two batches were produced, one with calcein present in the liposomes, and one without. Also an aqueous solution of PEG in 10 mM Hepes buffer was prepared. Both solutions were flushed with nitrogen for 10 min before liposomes were added and were subsequently transferred to a scintillation vial (total weight 5 g) . The resulting two phase system was vigorously vortexed for 60 s to create a water-in-water emulsion. Next, the emulsion was allowed to stabilize for 10-15 minutes (ambient conditions) , followed by the addition of TEMED (100 μl , 20% v/v, adjusted at pH 7 with 4 M HCl) and KPS (180 μl, 50 mg/ml) . This system was incubated from 30 min at 37°C to polymerize the methacrylate groups coupled to the dextran chains. Subsequently, the PEG phase was removed by multiple washing and centrifugation steps. The particle size and size distribution were measured with a laser light blocking technique (Accusizer™) . Different batches were prepared using varying liposome sizes (95, 184 and 370 nm) , microsphere water content (50- 70 %) and PEG/dex volume ratios (20-80) . The encapsulation efficiency of liposomes in the microspheres was defined as the amount of liposomes in the microspheres divided by the amount of liposomes added to the two phase system. The amount of liposomes in the microspheres was determined by measuring the phospholipid concentration in the microsphere pellet using the phosphate determination according to Rousser et al . (Lipids 5 (1970) 494-496). The encapsulation efficiency was very reproducible. For 5 independently prepared samples (formulation :dexMA DS 10, water content: 70%, volume ratio: 40, liposome size: 184 nm) , the encapsulation efficiency was 94.4%. In this case the liposomes were mixed with the dextran phase prior to addition to the PEG phase, in accordance with a preferred embodiment of the invention. When liposomes were added to the phase separated system, this resulted in an encapsulation efficiency of 88.1%. So, mixing the liposomes with the dextran phase first resulted in a higher encapsulation efficiency. The effects of liposome size, microsphere water content and PEG/dex volume ratio on the encapsulation efficiency were studied and are presented in Table 3. Table 3 Effect of water content, PEG/dextran volume ratio and liposome size on encapsulation efficiency (using DPPC: DPPG: Choi (10:1:10) liposomes and dexMA DS 10). Water Volume Liposome Encapsulation content (%) ratio size (nm) efficiency (%) 70 20 370 90 70 20 184 88 70 20 95 89 70 40 370 97 70 40 184 92 70 40 95 102 70 80 370 56 70 80 184 83 70 80 95 91 50 20 370 102 50 20 184 99 50 20 95 96 50 40 370 104 50 40 184 100 50 40 95 103 50 80 370 76 50 80 184 88 50 80 95 90 From this table 3 it appears that in most cases, the encapsulation efficiency is close to 100%. There seems no relation between the liposome size and the encapsulation efficiency (for any water content and/or volume ratio) . The encapsulation efficiency was expected to increase when larger liposomes were used, but apparently even the smallest liposomes were completely entrapped in the dextran matrix. A higher encapsulation efficiency was expected to a lower water content, because a lower water content results in smaller pores in the hydrogel. However, Table 3 shows that there is no relation between the water content and the encapsulation efficiency. For a volume ratio of 80, the encapsulation efficiency is slightly lower, because a large PEG/dex volume ratio results in a small volume of the dextran phase and therefore the liposomes can be less well encapsulated. Example 7 Example 6 was repeated using dexHEMA instead of dexMA as the crosslinkable polymer. All other steps were the same. Example 8 Example 6 was repeated using dexLactateHEMA instead of dexMA as the crosslinkable polymer. All other steps were the same . Example 9 The release of liposomes from the microspheres was measured by incubating the resuspended microspheres (in 10 ml Hepes buffer of desired pH) at 37°C. At desired time points and after centrifugation (10 min, 4500 rpm) , the complete supernatant was removed. The microsphere pellet was resuspended in 10 ml fresh buffer and put back at the roller bench. The amount of liposomes in the supernatant was determined by the phosphate determination according to Rousser et al . (Lipids 5 (1970) 494-496). The integrity of the released liposomes was studied using calcein containing liposomes and determining the fluorescence (λ ex =485, λ em =512 nm) of both the untreated supernatant and the supernatant treated with triton X-100. Due to the high concentration of calcein inside the liposomes, quenching occurs, resulting in a low fluorescent signal for intact liposomes. If calcein is released in the surrounding medium, the calcein is diluted resulting in a higher fluorescent signal. Treatment with triton X-100 results in destruction of the liposomes and the release of all calcein. The difference between the release of free calcein and the release of total calcein (after 4-S Φ 4-5 tn » CO β Φ TS X 3 oV> td CQ β Φ Ti -rl X 3 X Φ CQ CQ 4-J CO 0 o β Φ - l SH β 4J Xl CJ 0 EH 4-J φ rd φ A o ^ M SH r-i 4-S O (0 rd EH -H ε β Ss 4-S rl fd rH 4-5 0 & rd ε X X! rd . φ ε φ 0 X rd Φ CJ 4H tn ε CJ SH β 4-5 • 35 SH CO CQ H CQ X 4H © id Ti Φ rd -H Φ o 4-J © Φ 0 φ O Φ 4-J a 0 β 4-5 Φ Ti Φ CQ TS xi -H CQ CJ 4H XI -rl ti ft • ε β CQ -H β -H r-i Φ ti β 4-S 4-5 Φ fO O © 4-5 ft -H ^ — , 0 φ 0 φ > -H TJ fO 4-J 4H φ -H SH rd 4-5 4-S . -J fd rH ro Φ CQ CQ rl ε ■ H 3 4-S β X 3 β rd β O CO Z Φ Xl CD II X 0 Φ CJ -H cn T3 4J 0 Φ • i 4-5 - fc ■ H CJ -H J 4-5 H β Φ -H 4H β 4-5 a SH -H 4-S Φ CQ 4J CQ β TJ B ■ H P r-i U ■ ri XI 0 •— ' -H a ε 1 ^ CQ Φ 05 4H φ • ti Tl CQ ■ 23 a. ε CN 4H rH cn Φ rd CQ » SH 0 SH O φ β Φ CQ SH φ φ Φ 0 CQ Φ rd Φ Φ J β a 3 φ 4-5 •rl SH CD Φ Φ CQ Φ CQ r-i Φ Φ X! r-i ti rH 35 -H rH 4-S CQ rH ε φ Φ β ti ■U rd rd A β XI ε 4-5 4H φ Φ Φ TS β rd 0 TJ CQ XI -H Φ Φ ft Φ -H 0 4-J 0 rd rl CQ ϋ SH SH Φ Φ CO ■ H ft SH Φ Tl r- ε rH ϋ • H CO 4H 4J Φ rH rd fO ε ε 0 Φ X CQ Φ X! φ rd φ 3 -U 4H 0 0 SH Φ φ ε 03 XI 4-S a 4J H 0 4-5 4-1 0 ti X ti Tl rd 0 a Φ CQ ti 0 CJ Φ rd CQ -H rd ti 4J 4-1 PQ 0 TJ -H CQ 4J φ CQ 4-J 4J φ rd rH Xi • CJ rd .. 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X rO X rd Φ rl 4-J 0 ti -H 4H CQ ε ti CQ SH H rl 4-5 Pu CJ CO U 35 4-5 4-S J 4-S 35 ε rd Table 4 Liposome size and polydispersity index (PD) before encapsulation and after release from the microspheres. Before encapsulation After release Size (nm) PD Size (nm) PD 370 0.31 342 0 . 35 184 0.10 196 0 . 14 95 0.05 102 0 . 19 From Table 4, it is clear that the particle size after release is similar to the particle size before encapsulation in the microspheres. The effect of the liposome size on the release characteristics is presented in figures 11A and 11B for pH 7.2 and 8.0, respectively. From both figures it is clear that the liposome size does not affect the delay time. Furthermore, from Figures 11A and 11B it follows that the pulse length varies only little with particle size. In Figure 12, the effect of the PEG/dextran volume ratio on the release characteristics is presented. As expected, lower volume ratio results in a longer pulse, because a lower volume ratio results in a larger volume of the dextran phase. Therefore, more cross-links have to be degraded to release all liposomes, resulting in a longer pulse. The onset of the pulse was not affected by the volume ratio, because the pore size and the degradation rate is the same, irrespective of the volume of the dextran phase. The effect of water content on release is given in Figure 13A. Release over a longer period was observed. However, the lag-time did not increase for lower water contents, although the amount of initially released liposomes was smaller for the microspheres with the lower water contents. The above-mentioned effects of the water content were not observed when dexHEMA with a lower degree of substitution was used (Fig. 13B) . This can be explained by 5, the ratio between the time where 50% of the liposomes are released for both pH values are represented. Table 5 The theoretically expected and experimentally found effects of the pH on the release rate of the liposomes from dexHEMA microspheres. Water Degree of PEG/dex Liposome [OH-] 50% rel. content (%) substitution Vol .ratio size (nm) ratio ratio 70 8 40 370 4.9 4.2 70 8 40 184 4.9 4.2 70 8 40 95 4.9 4.2 70 8 20 184 4.9 5.3 70 8 80 184 4.9 4.1 50 8 40 184 4.9 5.3 70 5 40 184 4.9 2.5 50 5 40 184 4.9 2.5 The theoretically expected difference in release rate correspond well with the values found experimentally. The discrepancy observed from DS=5 again can be explained by swelling of the hydrogel.

Claims: Claims 1. A method for the preparation of microencapsulated colloidal systems, which method comprises the steps of: a) providing an aqueous mixture of i) a first phase comprising a water soluble crosslinkable polymer, ii) a second phase comprising a water soluble polymer which is incompatible in solution with the polymer in said first phase, and iii) colloidal systems to be suspended in said first phase; forming an emulsion of said first phase in said second phase, and b) forming microspheres in the emulsion by crosslinking at least part of said crosslinkable polymer, thus forming said microencapsulated colloidal systems. 2. The method of claim 1, in which step a) is carried out by forming a premixture by contacting said colloidal systems with said first phase, and subsequently contacting this premixture with said second phase. 3. The method of claim 1, in which said colloidal systems are selected from the group consisting of liposomes, iscoms, polyplexes, lipoplexes, nanoparticles, solid lipid particles in the colloidal size range, emulsions, and combinations thereof. 4. The method of claim 1, in which said crosslinkable polymer is a dextran polymer which comprises crosslinkable groups . 5. The method of claim 4, in which said crosslinkable groups are methacrylate groups. 6. The method of claim 5, in which said dextran polymer is selected from the group consisting of dexMA, dexHEMA and dexLactateHEMA. 7. The method of claim 6, in which the DS (degree of substitution) of said dextran polymer is from 2 to 30. 8. The method of claim 1, in which said polymer in the second phase is selected from the group consisting of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) . 9. The method of claim 1, in which colloidal systems are used which comprise an active ingredient, which active ingredient is preferably a protein drug. 10. Microspheres, at least 80 wt . % thereof having a particle diameter of between 100 nm and 100 μm, preferably between 5 and 15 μm, which microspheres are comprised of a degradable crosslinked polymer which encapsulates colloidal systems . 11. Microspheres according to claim 10, being free from organic solvent . 12. Microspheres according to claim 10, in which said colloidal systems comprise an active ingredient, which active ingredient is preferably a protein drug.

Inventors: FRANSSEN OKKE, HENNINK WILHELMUS EVERHARDUS, FRANSSEN, OKKE, HENNINK, WILHELMUS, EVERHARDUS
Applicants: FRANSSEN OKKE, HENNINK WILHELMUS EVERHARDUS, OCTOPLUS B V, FRANSSEN, OKKE, HENNINK, WILHELMUS, EVERHARDUS, OCTOPLUS B.V.
Application year: 2001
Main classification: A61K9
IPC codes: A61K9, A61K38, A61K47, B01J13