CA2153343A1 - Small diameter nanocapsules, process for their preparation and applications thereof - Google Patents
Small diameter nanocapsules, process for their preparation and applications thereofInfo
- Publication number
- CA2153343A1 CA2153343A1 CA002153343A CA2153343A CA2153343A1 CA 2153343 A1 CA2153343 A1 CA 2153343A1 CA 002153343 A CA002153343 A CA 002153343A CA 2153343 A CA2153343 A CA 2153343A CA 2153343 A1 CA2153343 A1 CA 2153343A1
- Authority
- CA
- Canada
- Prior art keywords
- nanocapsules
- phase
- nanocapsules according
- cyanoacrylate
- active agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
Abstract
Nanocapsules typically having a diameter in the range of 20-150 nm are provided and consist of a polymeric shell formed of a surface active poly(alkyl cyanoacrylate) material arranged in one or more layers.
The polymeric shell can be composed of a surface active poly(alkyl 2-cyanoacrylate) having general formula (I), wherein R is -CH2?CH2?mCH3, -CH2?CH2?mCOOH, -CH2?CH2?mCOOR', or -CH2CH2-O?CH2CH2-O?mR'; R' is -CH3, -CH2?CH2?mCH3, -C?CH3?2-CH2-C(CH3)3, or -O-Ar, m has a value of from 0 to 20; and n has a value of from 1 to 20, formed by the interfacial polymerization of self-arranged micelles of the cyanoacrylate monomers. The nanocapsules can be used to encapsulate a wide range of water soluble and water insoluble active agents in high yield for delivery to a target system or locus, for example drugs for use in therapy or prophylaxis.
The polymeric shell can be composed of a surface active poly(alkyl 2-cyanoacrylate) having general formula (I), wherein R is -CH2?CH2?mCH3, -CH2?CH2?mCOOH, -CH2?CH2?mCOOR', or -CH2CH2-O?CH2CH2-O?mR'; R' is -CH3, -CH2?CH2?mCH3, -C?CH3?2-CH2-C(CH3)3, or -O-Ar, m has a value of from 0 to 20; and n has a value of from 1 to 20, formed by the interfacial polymerization of self-arranged micelles of the cyanoacrylate monomers. The nanocapsules can be used to encapsulate a wide range of water soluble and water insoluble active agents in high yield for delivery to a target system or locus, for example drugs for use in therapy or prophylaxis.
Description
` ~4/15590 PCT/IE94/00001 21~3313 Descrip~ion Small diameler nanocapsules process for tlleir preparation and applications thereof Technical Fiekl Ihis invenlion relales lo small diame~er (20-150 nm) nanocapsules, a process for their prepara~ion and lhe use of the nanocapsules so prcpare(l for Ll)e delivery of active agents.
13ackgroun(1 Arl Nanocapsulcs are examples of nanoparticles which are used i~lter alic~ as drug carrier systems. NanoparLicles are either small solid spheres (nanospheres) or small capsules (nanocapsules) formed of a central cavity surrounded by a shell or wall.
Nanoparticles can be used to achieve controlled delivery of drugs and also to deliver drugs to specific target cells.
l hus, nanoparlicles are used illter alia to administer labile active agents or toxic anti-lumour agents to a subject. Conventionally, nanoparticles are administered by the intramuscular or intravenous route and are transporled into lhe epithelial cells, blood cells and liver cells by phagocylosis. Al~ernatively, the nanoparticles are degraded by chcmical and/or cnzylllalic proccsses in the blood.
Nanoparticlcs, sucll as poly(ethyl cyanoacrylate) particles, can be broken down by the Kupfrer cells of the liver resulting in release of the active agent.
The dislribulion and fate of nanoparticles in the body after administra~ion therelo depends on nanoparticle diameter. Small diameter nanoparticles (50-lO0 nm) are broken down by epithelial cells of the blood vessels. Middle size nanoparticles (100-400 nm) are ~ 94/15590 PCT/IE94100001 21~33~3 mainly brokel- down by blood cell~s. Nanoparticles with a diameter largel- lhall 300-800 nm are mainly broken down by the Kupffer cells of the liver. Super snlall diameler nanoparlicles (10-30 nm) are able to penetrate tlle blood-brain barrier and to deliver drugs into the brain.
Known types of nanopar~icles (Review Article; Couvreur, P. and Vauthier, C. (1991). Journal of Controlled Release 17, 187-198) include poly(bulyl cyanoacrylate) nanocapsules (Al Khouri Fallouh, N.
(1984); Pharm. I'h.l)., No. 207, Paris Xl) and poly(isobutyl cyanoacrylate) nanocap~sule~s (Al Khouri Fallouh, N. (1986);
International Journal of l'harmaceutics 28, 125-132). The latter paper describes a proce.s.s ror the formation of nanocapsules by a mechanism which is described as being probably ~lat of interfacial polymerisation resulting from lhe dispersioll of an alcoholic solution of isobutyl cyanoacrylate and oil in water. This process involves the use of two immiscible phasc~s and lhe nanocapsules so formed are oil-filled and can be used to entrap lipophilic substance~s. Only middle size nanocapsules with an average diameter of 200-300 nm can be obtained by the process described.
Damgé, C. c~ cll. (Diabetes (1988) 37, page 246) describe poly(alkyl cyanoacrylale) nanocapsules as a drug carrier for insulin.
l he rate of encapsulation of in~sulin was found to be 54.9%. The nanocapsules were prepared by the method of 1~l Khouri Fallouh N.
(1984) s~ anll as such the insulin was encapsulated in a lipophilic pha~se.
l here is a need for stable, aqueous- and non-aqueous-filled nanocapsules so as to extend the range of active substances that can be (lelivere(f by mean.s of such nanocapsules. There is also a need for slable small diameler nanocapsules capable of delivering active agents into target cell~s ~ administration to the human or ~nirrl7~1 body, including the vascular system and the brain.
EP-A 0 27~ 961 describes the preparation of middle size nanocapsules (100-400 nm) from dispersible colloidal systems. lt is ~ 94/15590 PCT/IE94/00001 21~33~3 indica~ed lhal a wide rallge of ~sub~stance~s (B) whieh are soluble or dispersible in a given ~solvent can be encap~sulated by the proeess deseribed. However, llle process as described will result in a eore of an organic solvent, an oily pl-ase or a particulate substanee. This will 5 limit the na~ure of ~he aclive agent ~hal can be eneapsulate(l. For example, many of Ihe organic solvents or solvent systems described would affeet Ihe ~stability of biologieally active agents, sueh as peptides and proleins, and would be likely to Iead to denaturation thereof and loss of pharmacological ac~ivily. l lle document does not deseribe the 10 iormation Or aqueous-rllled middle size nanoeapsules nor small diameter nanoeap~sules.
Olllel examples ol` microcap~sular drug carrier systems inelude lipo~some~s wllie}l are ~small pllo~spl~olipid ba~sed vesieles having an aqueou~s core. Lipo~somes obtailled by crosslinking of leeithin have 15 lipoidic wall~s ~struelurally related to those of biological membranes and as sueh have a ~shell defined by a molecular bi-layer.
Using leeilhin i~ is possible to syn~hesize small diameter vesieles (20-50 nm). However, liposomes are difficult to manufacture on an industrial scale. It is found that entrapped agents desorb very rapidly 20 from liposomes into the bloodstream, such that drug delivery to pha~oeytic cells i~s not achieved.
This desorp~ion of active agent and hence the lirnited stability of liposome~s hs due principally to the rapid hydrolysis thereof by blood enzyme~s.
Moreover~ the enLrapment levels of aclive agent achieved with liposome.s are low (see ~I Khoul-i ~allouh. N. (1986) sllpr-a).
Way.s of implovillg ~rug delivery, so as to aehieve better bioavailability and pharlllacokinetics are constantly being sought, e~specially for active agent~s which are subjeeted to rapid degradation following aclminkstratioll.
~ 94/15590 PC'rlIE94/00001 2t533~3 Oral adminis~ralion is one of ~he modes of administering drugs wl-ich has the grea~est clegree of patient compliance and thus ways are constantly being sougl-t Or formulating active agents for administration by the oral route whicl- it has not hitherto been possible to administer S by that route.
'~he low hydrolytic stability of liposomes means that they cannot be used for lhe delivery Or active agents by Lhe oral route. Enzymes of ~he gastrointeslinal lrac~ rapidly destroy liposomes following administration ~ tlle oral route with release of active agent, so that 10 uptake of active agent fronl the intestinal tract into the bloodstream is nol reali~.e~.
Di~clo~sure of Invention The invenlion provides nanocapsules comprising a polymeric shell formed of a surface active poly(alkyl cyanoacrylate) material 15 arranged in one or more layers.
The nanocapsules according to the invention are stable and can be used to entrap effective amounts of an active agent. lt is possible to achieve a degree Or encapsulation of 75% or higher with the nanocapsules according to the invention.
E3y polymer herein as regards the polymeric shell is meant any suitable polymer according to the l.U.P.A.C. definition of polymer.
The polymeric shell of the nanocapsules according to the invention is made up of polymer chains typically of the order of 10 or more monomer units. The polymer shell formed has the ordered arrangement of a mono-, bi-, ~ri-, or polymolecular layer typical of a liposorne.
Preferably, the nanocapsules in accordance with the invention have a ~iameter in tl-e range 20-150 nm.
-~ 94/15590 PCTIIE94/00001 21533~3 l he size of the nallocapsules is determined by the type of monomer used and by ttle melhod of preparation as hereinafter described.
l`he presence of one or more ordered layer(s) in the polymeric 5 shell follows from lhe size of the nanocapsules produced and may be confirmed by electron diffractioll.
The nanocapsules according to the invention are primarily intended for use in tlle delivery of active agents to the human or animal body, includillg delivery for the purposes of medical diagnosis 10 involving imagil~g. However, lhe nanocapsules according to the inventioll are not limiled to such use and will also find application in agriculture, in cosmetics for delivery of a wide variety of active agents including fragrances, ~hc food industry and other areas of technology to which their properties are adapted to provide a desired effect. For 15 example, the nanocapsules according to the invention are ideally suited for the encapsulation and subsequent delivery of systemic fungicides, herbicides and pestici(les alld plant growth controlling agents to plants.
Thus, typically an active agent is contained in the aqueous- or non-aqueous phase contained in the core.
An especially preferred polymeric material for the nanocapsular shell is a poly(alkyl cyanoacrylate) material, more especially a surface active poly(alkyl 2-cyanoacrylate) having the general formula:
/CN
COOR "
~ 94/15590 PCT/IE94/00001 6 21~3393 wherein R i s --CH2 t CH2~mC~ 3 --CH2 ~ CH2~m COOH, ~ CH2 ~CH2tmcOoR, or--CH2CH2--0~ CH2CH2 ~ ~m R;
R' is--CH3,--CH~ ~CH2~l,CH3.--C~ CH33~ CH2-C(CH3)3~ or --O--Ar;
m has a value Or from () to 20; and n has a value of from I to 20 According to a preferred melhod, the nanocapsules are formed 5 by in~erfacial polymerisation of self-arranged micelles of surface active cyanoacrylate monomcrs in an aqueous phase as hereinafter described The active agent encapsulated in ~he nanocapsules according to the invention is any water soluble or water insoluble active agent, including naturally occurring substances and synthetic analogues 1 0 thereof Given that the active agent is dissolved or dispersed in an aqueous or non-aqueous phase in the core of the nanocapsule, the stability thereof is maximised Especially preferred active agents for encapsulation in the 15 nanocapsules according lo the invention are water soluble active agents Such preferred active agents include arnino acids, peptides and polypepti~les Such aclive agents include hormones, hormone release factors, cytokines, encephalins, blood factors and products including enzymes and anlibodies, and other active agents which are susceptible 20 to degradation and/or modification by proteolytic and other enzymes before exerting lheir effect, especially if administered by the oral route. 'I he latter type of active agents also includes anti-tumour agents, antibiotics~ opiates ~such as apomorphine, dopamine, serotonin and ` ~94/15590 PCTIE:94100001 21~33~3 other agents active on tlle cenlral nervous system, and steroid hormones such as proge~sterorle and te~slosterone.
When the aclive agent is an antibody, the antibody can be a monoclonal or polyclonal anlibody.
'I he nanocapsules according to the invention are also especially suitable for the encapsulation and subsequent delivery of immunomodulating agen~s, for example, cyclosporin.
l`he nanoc.lpsulcs according to the invention can also be used to encapsulate variou.s VaCCillC~s.
lt will be appl-cciatcd that the nanocapsules according to the invention can inclea~se lhe bioavailability and efficacy of a wide range of water soluble ac(ive agents by protecting said agents from premature degradation in the gaslrointestinal tract and Lhe blood and allowing for a sustained release thereof~
The invention also provides an active agent delivery system comprising nanocapsules as hereinbefore described.
The nanocapsules according to the invention are stable and release their contents on degradation following ~(lministration to the target system or locus.
The nanocapsules according to the invention when intended to deliver an active agent for use in therapy or prophylaxis may be administered orally, parenterally or topically to the human or animal body. Following oral administration the nanocapsules traverse the gut wall and are taken up into the blood stream and the product is released on degradation of lhe nanocapsule shell or wall.
The nanocapsules are useful in delivering active agents to the blood stream by ~he oral rou~e lhat are not normally suitable for administration by ~his rou~e in traditional conventional pharmaceutical ` ~94/15590 PCT/IE94/00001 21~33~3 fortnulations. I he cncapsulated product is protected from the harsh condilions of lhe gul, SUC}l that a significantly greater proportion of active agent is delivere(l ~o t}-e bloodstream than would be possible by simple oral administration of the non-encapsulated active agent. For 5 example, insulin, a prolein, is normally given by intramuscular injection. If given orally i~ is normally degraded by the normal diges~ive processes of ~l~e gut an(l only a very small and variable proportion finds i~s way in~o ~lle bloodstream. lnsulin encapsulated by the met~lod according ~o ~he invention can be given orally wi~h minimal 10 lo~s~s of pharmacological effec~ Thus, there are major benefits for the patien~ botll in ~CI lllS of reducing stress and increasing convenience Suitable formulatiolls of the nanocapsules according to the invention for adlllinistration by the oral route include capsules, dragées, elixins, granules, lozenges, pellets, powders, suspensions and 15 tablel~s. In the ca~se of tablets care should be taken that the tabletting ~echnique doe~s no~ Icad ~o any disruption of ~he nanocapsules and alteration of their release properties. Such tablets can be formulatecl for rapid disintegration in the gastric and/or intestinal juices, if required or, alternatively, coated so as to further delay the release of 20 the active agent The nanocapsules can also be formulated as solutions or suspensions for injection intramuscularly, intravenously and subcutaneously 1~ is also possible to formulate the nanocapsules accordillg to tl)c invention in liquid form for administration by 25 perfusion.
Further types of formulations according to the invention include nasal formulations, ocular agents, including slow release implants containing the nanocapsules, pessaries, suppositories, lozenges coated on one surl;.lce witll a bioadhesive for use in the buccal cavity or 30 formulations for adlllinis~erillg an active agent sublingually~
` ~4/15590 PCT/IE94/00001 21~33~3 'I`lle nanocap.sllle~s will gellerally be formulated in unit dosage lorlll for a~lminislr.llion or applicalion in an amount and for a time pre~scribe~ by all a(len~ing pllysician.
A preferre(l me~llotl for preparing lhe nanocapsules aeeording to 5 the invention comprise~s inlerracial polymerisation of a surfaee aetive cyanoacrylale mollolllel in the form of a colloidal solution eomposed Or self-arranged micelle~s in an aqueous medium under polymerisation inilialing condilioll.s. Colloidal parlicles of an aclive agent to be eneapsulaled may serve a~ the initia~or of polymerisa~ion. However, 10 polymeri.salion can also be spontaneou~s. The aqueous medium is preferably compo.sed ol` a lwo phase aqueou~s system.
A single or mollo-pllase aqueous system for use in aeeordanee with the invenlion i~s typically a solution physiologieally isotonic in s~reng~h and eomprises wa~er or an aqueous solution of one or more ]5 water-soluble polymer~s and/or one or more water soluble salts.
~ two pha~se aqueou~s system for u~se in aceordanee with the invention preferably comprises an aqueous eolloidal solution of two or more water soluble imtniseible polymers. Sueh water soluble immiseible polylllel-~s are known (see for example, Alberdsson, P.A.
20 "l'artition of eell par~ieles and maeromolecules", Wiley, lnternational Scientific N.Y. (1971) pp 30-37).
lhe polymers are selected primarily on the basis of their eompalibilily ancl den.sity. As regards the former criterion, there should be little or no affinity between the polymers, sueh that they do 25 not form aggregate~s or interaet unfavourably in solution.
'1 he followill~ are examples of suitable combinations of polymer~s:
DexLran sulphate an(l methylcellulose L~extran sulpl-ale and polyethyleneglyeol l. ~
` 1~94/1~590 PCT11E94100001 2I~3313 l)exlran sulpllale antl polyvinylalcohol Dielhylaminoelllyldextran and polyelhyleneglycol Dextran and polyvinylalcohol Dextlan and melhylcellulose Dextran and polyethyleneglycol Dextran and T~icoll (l~icoll is a Trade Mark) Dextran and oxypropyldextran L)extran and a mixed polymer of ethylene oxide and propylene glycol sucll as l'lurollic (I'luronic is a Trade Mark) Dextran alld polypropyleneglycol Oxypropyldextran and polyethyleneglycol Sodium carboxymelhyldextran and polyvinylpyrrolidone Dex~ran and chi~osan Dexlran and dextran sodium sulphate The formalion of a stable ~wo phase system also depends on the concentralions Or tllc rcspeclive polymers in the solution. lf the concentration of the polymers is below a critical level, then the two aqueous polymers will not separate into layers. The behaviour and characterislics Or difrerent polymers in combination must be 20 determined empirically (see Alberdsson, P.A. (1971) sl-pra).
The polymerisalion initiating conditions preferably involve the use of an initiator Or anionic polymerisation, which initiator is located within colloidal par~icles of a discrete phase or within a continuous phase. Examples of anionic polymerisation initiators include substances 25 containing nucleophilic groups such as, for example, amines and thiols.
Generally, the conlinuous phase will be present in a large excess relative to the discrcte phase, for example in a ratio of 100:1-50:1.
The interfacial micelles of cyanoacrylate monomer preferably sel~-arrange on the surface of colloidal particles of a discrete phase or 30 witllin solutioll. To provide a cyanoacrylate monomer having the ability lo self-arrangc in an aqueous medium to form micelles surface ` ~4/15590 PCT/E94100001 ~1S33~
active cyanoacrylic monomers of the type hereinabove defined are used.
In order to provi(le cyanoacrylate monomers having surface active propcrlies, hydrophilic or hydrophobic radicals are introduced 5 into the cyanoacrylate molecule as hereinafter described.
The synthesi~ of surface active cyanoacrylate monomers for use in accordance wiLh tlle invention is illustra~ed by the following preparation Example~s.
Suitablc aniollic typc surrace active alkyl 2-cyanoacrylates having 10 an ionogenic moiely in the ester radical have ~he general formula:
/CN
C~12=C o Il--O~CH2tnC~
O OH
wherein n has a value of from I to 20.
Suitable nonionic surface active alkyl 2-cyanoacrylates having a 15 hydrophilic moiety in the ester radical have one of the following formulae:
/CN
i) CH2=C ~0 C--O ~ CH2tn C
OR
` ~94tl5590 PCT/IE94/00001 12 21S334~
wherein R is--CH3,--CH2~CH2~mCH3~--CtCH3t2--CH2(CH3)3- or --O--Ar; an~
m and n each has a value of from I to 20;
CN
ii) CH2--C CH3 C~H3 C--O ~ CH2CH2O ~¢--CH2--I--CH3 wherein n has a value Or Irom I to 20; and /CN
iii) Cl~2=C
C--O ( CH2 )--CH3 wherein n has a value of from 0 lo 20.
The principal stages in the encapsulation process are as follows:
5 Stage 1: The preparation of a continuous phase.
Stage 2: Polymerisation of a cyanoacrylate monomer to form capsules.
.n~rlpsul;ltion using ;- mono-ph;l~e queous system ln Stage 1, an aqueous solution is formed for use as a polymerisation medium for obtaining capsules. Preferably, an 10 isoosmotic solution con~aining water soluble polymer and corresponding salts is used. An inhibitor of polymerisation, preferably cyanoacrylic acid, and a water soluble active substance are added to form a solution which is physiologically isotonic in strength.
` ~)4/15590 PCT/IE94/00001 In Stage 2. a surface active ester of cyanoacrylic acid is added wilh vigorous stirring, prcferably using sonication. Micelles of monomer are formed in a continuous phase and then the monomer polymerises to form a solid shell of polymer. To increase the rate of 5 polymerisa~ion an initiator having a basic nature sucll as hydroxyl ion or heating to a temperature of up to 60C is used. The active substance is encapsulated following polymerisation of the monomer to form solid micelles due to the difference in hydropl~ilicity inside and outside the micelles. Prererably the solid shell is composed of a mono- or bi-10 molecular laycr of polylllcr.
Encapsulation U.Sill~ a two pha.se sy.stem Encapsulation u~sing a colloidal .solution of a water insoluble ac~ivesubstance The preparation of the continuous phase in the Stage 1, is similar 15 to that for a mono phase aqueous system hereinabove described.
However, the active agent is added in the form of a solution in an organic solvent, preferably an alcohol, or in the form of a suitable aqueous solution containing one or more solubiliser(s) or other additives.
ln Stage 2, the size of colloid particles of the active agent is determined before addition of the monomer, and the colloid solution is sonicated if necessary. The surface active cyanoacrylate monomer is added with stirring an(l tlle micelles of monomer are formed on the surface of colloid particle~s of active substance. The solid shell is formed on the surface of colloid particles following polymerisation of the monomer ~o form a polymolecular layer of polymer. The surface of the colloid parlicles serves as the initiator of polymerisation while a bulk phase serve~s as a polymerisation inhibitor.
` ~94/1!;590 PCT/IE94/00001 21~33~3 Encapsulation using a two phase aqueous solution ln Stage 1, a two phase aqueous system is formed containing an aqueous solution of two or more immiscible water soluble polymers capable of forming such a lwo phase systeni as follows. The polymers 5 selectetl are dissolve~ in water, whereupon they settle into two layers, following equilibration of the respective polymers. This separation occurs primarily because the polymers are of different densities. If require(l, a further polymer which partitions selectively into one or other of the layers may be added during this stage, said further 10 polymer having ~he capabilily to selectively concentrate a target active agent ~o be a(l~ed in sta~e 2 and which it is desired to encapsulate in either one or the o~her phase of the two phase system.
The rate or time required for the sep~ration of the layers depends on the choice of the individual polymers. Left to gravity 15 alone, the separalion can take from several hours to several days.
Separation can be accelerated by centrifugation.
After separation, the two aqueous solutions, upper and lower, are decanted into separa~e flasks. At this point, two stable systems have been created in whicll the upper phase is equilibrated with lower phase 20 and l~ice l~er.~a. Both phases are used in the creation of an emulsion in Stage 2.
Stage 2:
In lhis stage~ an emulsion is formed between the two separated phases described above. Typically, the emulsion formed contains the 25 two phases in a ra~io of ~he order of 100:1. On emulsification, small droplets of the minor component form a discrete phase dispersed within a bulk or continuous phase of the major component which is present in excess. ~s both components in the emulsion have been mutually equilibrated in Stage l, the droplets are relatively stable in the 30 mixture and thus an emulsion can be formed.
~ 94/15~90 PCT/IE94100001 21~33~3 The emulsion is formed by vigorous agitation such as that achieved by means Or sonication or vortex mixing. The size of the droplets forming the discrete phase is primarily controlled by the degree and rate of agitation.
'I he active a~ent to be encapsula~ed is included in the emulsification proces.s. 'I he choice of upper or lower phase (created in Stage I ) to form the minor component in the emulsion is determined by ~he physical and chemical properlies of the active agent. Indeed, such properties also inlluence the choice of polymers used in Stage 1. The droplets within ~he emulsion become encapsulated by the alkyl 2-cyanoacrylate added in ~he next stage - Stage 3. Accordingly, it is desirable to selectively concentrate the active agent inside the droplets.
Thus the choice of upper or lower phase to form ~he droplets in the emulsion is primarily determined by the affinity of the active agent for IS the respective phases.
The method also allows for an initiator of polymerisation to be concentrated inside the droplets. In some instances, this may be the active agent itself. If this is not an initiator, however, this must also be added at this stage.
Stage 3:
In this stage, an alkyl 2-cyanoacrylate is added and the droplets are encapsulated following polymerisation at the droplet surface. Alkyl 2-cyanoacrylatcs polymerise on contact with an initiator of polymerisation. Nucleophilic chemical groups are good initiators of polymerisation. Conversely, acids, particularly strong acids, inhibit the polymerisation process. ln the emulsion created in Stage 2, polymerisation in the bulk phase or continuous phase is inhibited by a low pH. This effect is reversed when the monomers encounter the initiator at the surface of the droplet, resulting in encapsulation by polymerisation of the droplet which includes the active agent.
` 9~94/15590 PCTlIE94tOOOOl 16 21~33~
Apart from the degree of agitation a number of other factors delermine the size Or tlle nanocapsules according to the invention. ln general, the more rapid thc rate of polymerisation, the lesser the degree of control over polymerisation and the greater the size of the 5 nanocapsules formed.
Furthermore, the rale of polymerisation is inversely proportional lo the size of the alkyl group in the alkyl cyanoacrylate monomer, so that the larger the alkyl group, the slower the polymerisation and hence the smaller the nanocapsules formed.
Also the lower the pH of the aqueous solution, the slower the rate of polymerisation and lhe smaller the size of the nanocapsules formed.
The rate of polymerisation is primarily controlled by pH and the size of the ester (alkyl) group~s.
Best Mode~s for Carrying Out the Invention The invention will be further illustrated by the following Examples.
In the following Examples the method according to the invention is exemplified by lhe encapsulation of a dye (DiI) and the peptide type hormone oxytocin.
Example I
2-Cyanoacrylic acid e~sler of polyethylene glycol 4-tert-octylphenyl ether A 500 ml flask was fitted with mechanical stirrer, thermometer, argon and sulphur dioxide inlet adaptors, dosing funnel protected with 25 a drying tube, and Liebig condenser arranged for distillation. The flask was charged with 250 ml of anhydrous toluene, and 1 g of 2-cyanoacrylic acid was added to the boiling solvent with stirring and ~ ~94/15590 PCTtIE94/00001 21S33~3 sparging wi~h argoln 2U ml Or toluene/waler azeotrope was removed by dislillation antl 2.2 g Or phosphorus penlachloride dissolved in S0 ml of dry benzel~e was then added dropwise wiLh stirring and constant removal of benzelle by di~slillaliol~ e mixture was stirred under reflux for I llour and Lhen sparged with sulphur dioxide, while 100 ml of toluene containing by-product phosphorus oxychloride was distilled Orr to leave a resi(lual colourless solu~ion Or 2-cyanoacryloyl chloride in toluene. ~ solu~ion of 7.4 g of Triton-X100 (Triton is a Tradle Mark) and 0.5 g of hydroquinone in 50 ml of benzene was then added clropwi.se to l}le 2-cyalloacryloyl chloride solution with stirring and constant removal Or benzene by distillatioll. l he mixture was refluxed during I llour, coolcd, an(l solvent was dislilled off in vacuum to give 8.1 g of a colourless oil which was washed with hot hexane to give 7.8 g of tl-e 2-cyanoacrylale ester of polyethylene glycol 4-tert-octylphenyl elher. Elemen~al Analysis Calculated for C3gH63NO2: C
67.35%, H 9.30%, N 2.07%, Found C 66.7%, H 9.6%, N 1.9%, lH
NMR in 1: I C6D(,: (CD ~)2CO 0.75 (9H, s, (CH3)3C-~, 1.37 (6H, s, (CH3)2C-), 1.77 (2H, s, CH2), 3.62 (m, CH2O-), 3.98 (t, J = 4 Hz, CH2O-), ~.13 (t, J = 5 ~Iz, CH2O-), 4.43 (m, 2H, CH2OCO-), 6.08 (s, 1H, ~-C=C-), 6.67 (~. I H, H-C=C-), 6.88 and 7.33 (2d, each 2H, A2B2, J = 7.2 Hz, aryl) ppm.
Example 2 2'-Carboxyethyl 2-cyanoacrylate 9.~ g of 2-cyanoacrylic acid, 0.2 g of 4-toluenesulphonic acid and 0.1 g of hydroquinone were dissolved in 250 ml of anhydrous benzene in a S00 ml flask which had previously been washed with 10 %
sulphuric acid and dried USillg acetone, and which was fitted with a stirrer, a lhermometer~ sulphur dioxide and argon inlet adaptors, a dosing funnel and a Liebig condenser arranged for distillation. The solution was sparged wi~h sulphur dioxide and brought to reflux when a suspension of 9.9 g Or 3-hydroxypropionic acid in 200 ml of benzene was added dropwise wilh continuous removal of benzene-water azeotrope by disLillalioll. rhe mixture was heated with stirring and ~ 94/1559û PCTIIE94/00001 21~3q~
sparging with sulphur dioxide unlil the benzene-water azeotrope ceased to appear, and was then refluxed for a furlher 30 minutes. The volume was reduced to 100 ml by removal of solvent by distillation. The residual colourless solution was cooled~ filtered, and diluted with 500 5 ml of heptane to give 8.5 g of a solid which was collected. The solid was recrystallised from 1: I benzene: heptane which had been satura~ed wi~h sulphur dioxide lo yield 6.51 g of 2'-carboxyethyl 2-cyanoacrylate.
Example 3 Preparatioll of hexadecyl 2-cyanoacrylate lnlo a 0.5 litre flask fitted with mechanical stirrer, thermometer, argon inlet adaplor wilh a device for admitting a stream of gas under lhe surface of tlle reaclion mixture, a dosing funnel protected with a Drierite drying tube, a Liebig condenser provided with a vacuum distillation adaptor and a receiver flask connected to a vacuum flask was chargetl 0.98 g (0.01 mole) 2-cyanoacrylic acid, S0 mg methylhydroquinone, 200 ml dry benzene and 100 ml dry toluene. A
solution of 2.08 g (0.01 mole) phosphorus pentachloride in 50 ml of dry toluene was charged into the dosing funnel. While sparging with dry argon and stirring under reflux the phosphorus pentachloride solution was added dropwise. Following completion of the addition the reac~ion mixture was boiled for 15 minutes following which the reflux condenser was substituted by a Liebig condenser with a receiver and a calcium chloride drying tube and 200 ml of solvent were distilled off.
At this point 2.42 g (0.01 mole) n-hexadecyl alcohol in 50 rnl dry benzene was added from the dosing funnel while refluxing and stirring and sparging with dry argon. ~ollowing addition of the alcohol the mixture was boiled for one hour and then the solvent was distilled off to give 50 ml ren-aining which was cooled to 5C and left overnight (17 hours), following which crystals of 2-cyanoacrylic acid had fallen out WhiCIl were fillered off. The volatiles were removed by distillation under vacuum and the remaining solid recrystallised from hexane to give 1.57 g n-hexadecyl 2-cyanoacrylate (49% yield) solid; m.p. Sl-` \~94/15590 PCT/IE94/00001 ,9 21~33q3 3~C [Elemental Analysis Calculated for C2()H35NO2 C = 74.8, H =
10.9, N = 4.4, Found C = 73.5, H = 11.1, N = 4.1].
lH NMR ~6.24 (IH, s, CH~=C-), 5.38 (IH, s, CHb=C-), 3.89 (2H, t, J=5.8Hz, -CH2OCO-), 1.32 (281-1, m, (CH2)l4-), 0.91 (3H, t, -CH3) ppm. 13C NMR C ~13.62 CH~. 22.29 CH~CH2~ 31.56 CH3CH2CH2, 29.0(CH2)1(), 25.27 CH~ (CH2)12 CH2, 27.95 (CH~,(CH2)1~CH2), 65.98 CH3 (CH2)14 CH2O, 113.85 C, 115.99 CN, 159.78 C=O.
Example ~
l~nCapSUIalion itl a mono-phase system Preparation of.self-arranged poly(2'-carboxyethyl 2-cyanoacrylate) nanocap.sulc~s in a mono-phase aqueous medium.
A coolecl sonicalion reaction vessel was filled with 50 ml of a solution of 50 mg of citric acid (citric acid monohydrate obtained from Belgorodsky Plant of Citric Acid, Belgorod, Russia) in isoosmotic I S dextran-based plasma ~ubs~itule "Polyglukin" (Polyglukin is a Trade Mark of Krasnovarsky 1'1ant of Medical Preparation). The mixture was cooled and titraled by H3PO4 to a pH of 2.5-3.2. Approximately 0.3 g of 2'-carboxyethyl 2-cyanoacrylate (prepared in Exarnple 2) was added in portions wil}l continuous sonication and cooling of the reaction vessel to provide a reaction temperature not higher than 30C.
When the solution became cloudy adding of 2'-carboxyethyl 2-cyanoacrylale was sloppcd and the mixture was sonicated for 30 min.
with continuous cooling. The sonication should be stopped and the mixture cooled in the case of spontaneous heating. The nanocapsules obtained were sized by a Coulter Counter. ln the remaining Examples, the nanocapsules produccd were sized in the same way. The mean diameler Or nanocap~slllc.s obtained wa~s 25 nm. 95% of nanocapsule~
had a size of 25 nn~ .s~nLlard deviation 20 nm.
~94115590 PCT/IE94/00001 21~33~3 Example 5 Encapsulation of oxytocin in self-arran~ed nanocapsules f~rmed from the 2-cyanoacrvlic acid ester of polyethylene glYcol 4-tert-octylphenyl e~her using a mono-phase system A cooled sonicalion reaction vessel was filled with 50 ml of a solution of 50 mg of cilric acid in an isoosmotic salt-based solution.
The mixture was cooled an(l titrated wi~h phosphoric acid to a pH of 2.5-3.2. 2500 U of oxytocin was dissolved with stirring using a magnelic stirrer an(l 0.5 g of surface active 2-cyanoacrylic acid ester of polyethylelle glycol ~-tcrl-octylphenyl e~her monomer prepared in Example I was a~de(l dlopwise with conlinuous sonication and cooling of the reaction vessel to provide a reaction temperature not higher than 30"C. When the solution became cloudy adding of monomer was stopped and the mixture was sonicated for 15 min. with continuous cooling. The sonication should be stopped and the mixture cooled in the case of overheating. After this time the suspension is transferred to a magnetic stirrer and stirred for 24 hours. The pH is then adjusted to 7.2-7.4 by the addition of IN NaOH with continuous stirring. The nanocapsules produced were sized. The mean diameter of nanocapsules obtained was 48 nm. 95% of nanocapsules had a size of 48-49 nm, standard deviation 25 nm.
~xample 6 Encapsula~ion Or Dil in self-arranged nanocapsules formed from poly(hexadecvl 2-cyanoacrylate) In this Example. the method according to the invention is used to encapsulate the fluorescen~ dye named DiI (supplied by Molecular Probes Inc.. Eugene. Oregon, U.S.A.) useful for staining living cells in cell biology. Dil is insoluble in water and is a lipophylic substance accessible in tlle form of a solution in alcohol. Dil can be encapsulated 30 by the use of a s~rong lipophilic cyanoacrylate monomer in a mono-phase aqueous-organic medium.
~ 94/15590 PCT/IE94/00001 21~3313 A cooled .sonicalion reaclion vessel was rllled Wil}1 25 ml of citric buffer ~solution at pH=7.2-7.4 and 20 ml of alcohol containing I mg Dil. llle mixture was cooled with stirring using a magnetic stirrer and after complete di~ssolution of Dil the presence of colloidal micelles was 5 determined by a Coulter Coun~er. Further alcohol was added if necessary. I ml of alcol1ol solution containing 0.1 g of hexadecyl 2-cyanoacryla~e prepared in Example 3 was added dropwise with con~inuous sonica~ion alld cooling of Lhe reaction vessel to provide a re~ction temperalure not greatcr tllan 60C The suspension was 10 sonicaled for 30 mill~ willl continuous cooling. The sonication should be stopped and ~he mix~urc cooled in the case of overheating. Aher lhis time ~he suspen~sioll was transferred to a magnetic stirrer and stirred for 24 hours~ I he pH was then controlled and adjusted to 7.2-7.4 by lhe addition of I N NaOH with con~inuous stirring if necessary.
15 The nanocapsules produced were sized. The mean diameter of the nanocapsules oblained was 37 nm. 95% of the nanocapsules had a size of 37-38 nm standard deviation 25 nm. To estimate the yield of encapsulation the suspension was centrifuged at 45000 r.p.m. for 4 hour~ and ~he absorbance of the solution was determined by UV
20 spectro~scopy. Ihc es~ ated yield of encapsulation is 82%.
Example 7 Encapsulation of DiI in self-arranged nanocapsules formed from the 2-cyanoacrylic acid ester of polyethylene glycol 4-tert-octylphenyl ether In this Example a two phase aqueous system is used to 25 encapsula~e Dil. Under the appropriate conditions Dil is able to form an aqueous colloidal solulion.
A cooled sonicalion reaction vessel was filled with a 50 ml solution of 50 mg of citric acid in an isoosmotic salt-based solution.
The mixture was cooled and titrated with H3PO4 to a pH of 2.5-3.2.
30 mg of Dil in I ml of alcohol was added with sonication. The size of the micelles formed was determined by the use of a Coulter Counter and the mixture was sonica~ed until micelles with a size of 25-40 nm ` ~O4/15590 PCTIIE94/00001 22 21S33~3 were obtained. I llen 0.1 g Or the surface active 2-cyanoacrylic acid ester of polyelhylene glycol 4-tert-octylphenyl ether monomer prepared in Example I was added dropwise wi~h continuous sonication and cooling of the reaction vessel to provide a reaction temperature not greater than 30"C. The mixture was sonicated for 40 min. Sonication should be stopped and ~he mixture cooled in the case of overheating.
After tnis time the suspension was transferred to a magnetic stirrer and stirred for 24 hours. The pH was then adjusted to 7.2-7.4 by the addition of lN NaOH wilh continuous stirring. The nanocapsules produced were sized. The mean diameter of nanocapsules obtained was 115 nm. 95% of nanocapsules had a size of 115-116 nm, standard ~evia~ion 65 nm.
Example 8 Ençapsulation of Dil in self-arran~ed nanocapsules formed from ~he 2-cyanoacrylic acid es~çr of polyethylene glycol 4-tert-octylphenyl ether ln this Example, Dil was encapsulated using an aqueous colloidal solution of two immiscible aqueous soluble polymers exemplified by the use of a dextranlpolyethylene glycol (PEG) two phase system.
Preparation of the two phase system:
Dextran (10 g) and PEG (1.14 g) (supplied by Schuchardt, Munich, l~edcral Republic of Germany) were dissolved in 50 ml of water by mixing and heating to 80C. After cooling 50 mg of citric acid and 0.2 ml of H3PO4 were added. If required, further H3PO4 was 2S added to adjust tne pH to 2.5-3. The mixture was then allowed to stand and the layers formed separated in a separatory funnel.
Preparation of colloidal solution and polymerisation:
I mg Dil dissolved in I ml of alcohol was added dropwise to 1 ml of upper phasc (primarily PEG). The solution obtained was added ` ~94115590 PCT/IE94/00001 23 21~33~3 to 200 ml of lower phase (primarily dextran). The rnixture was then placed in a cooled sonication reaction vessel and was sonicated with continuous cooling to provide a droplet size of 60-80 nm. The size of droplets was determined by the use of a Coulter Counter as before.
13ackgroun(1 Arl Nanocapsulcs are examples of nanoparticles which are used i~lter alic~ as drug carrier systems. NanoparLicles are either small solid spheres (nanospheres) or small capsules (nanocapsules) formed of a central cavity surrounded by a shell or wall.
Nanoparticles can be used to achieve controlled delivery of drugs and also to deliver drugs to specific target cells.
l hus, nanoparlicles are used illter alia to administer labile active agents or toxic anti-lumour agents to a subject. Conventionally, nanoparticles are administered by the intramuscular or intravenous route and are transporled into lhe epithelial cells, blood cells and liver cells by phagocylosis. Al~ernatively, the nanoparticles are degraded by chcmical and/or cnzylllalic proccsses in the blood.
Nanoparticlcs, sucll as poly(ethyl cyanoacrylate) particles, can be broken down by the Kupfrer cells of the liver resulting in release of the active agent.
The dislribulion and fate of nanoparticles in the body after administra~ion therelo depends on nanoparticle diameter. Small diameter nanoparticles (50-lO0 nm) are broken down by epithelial cells of the blood vessels. Middle size nanoparticles (100-400 nm) are ~ 94/15590 PCT/IE94100001 21~33~3 mainly brokel- down by blood cell~s. Nanoparticles with a diameter largel- lhall 300-800 nm are mainly broken down by the Kupffer cells of the liver. Super snlall diameler nanoparlicles (10-30 nm) are able to penetrate tlle blood-brain barrier and to deliver drugs into the brain.
Known types of nanopar~icles (Review Article; Couvreur, P. and Vauthier, C. (1991). Journal of Controlled Release 17, 187-198) include poly(bulyl cyanoacrylate) nanocapsules (Al Khouri Fallouh, N.
(1984); Pharm. I'h.l)., No. 207, Paris Xl) and poly(isobutyl cyanoacrylate) nanocap~sule~s (Al Khouri Fallouh, N. (1986);
International Journal of l'harmaceutics 28, 125-132). The latter paper describes a proce.s.s ror the formation of nanocapsules by a mechanism which is described as being probably ~lat of interfacial polymerisation resulting from lhe dispersioll of an alcoholic solution of isobutyl cyanoacrylate and oil in water. This process involves the use of two immiscible phasc~s and lhe nanocapsules so formed are oil-filled and can be used to entrap lipophilic substance~s. Only middle size nanocapsules with an average diameter of 200-300 nm can be obtained by the process described.
Damgé, C. c~ cll. (Diabetes (1988) 37, page 246) describe poly(alkyl cyanoacrylale) nanocapsules as a drug carrier for insulin.
l he rate of encapsulation of in~sulin was found to be 54.9%. The nanocapsules were prepared by the method of 1~l Khouri Fallouh N.
(1984) s~ anll as such the insulin was encapsulated in a lipophilic pha~se.
l here is a need for stable, aqueous- and non-aqueous-filled nanocapsules so as to extend the range of active substances that can be (lelivere(f by mean.s of such nanocapsules. There is also a need for slable small diameler nanocapsules capable of delivering active agents into target cell~s ~ administration to the human or ~nirrl7~1 body, including the vascular system and the brain.
EP-A 0 27~ 961 describes the preparation of middle size nanocapsules (100-400 nm) from dispersible colloidal systems. lt is ~ 94/15590 PCT/IE94/00001 21~33~3 indica~ed lhal a wide rallge of ~sub~stance~s (B) whieh are soluble or dispersible in a given ~solvent can be encap~sulated by the proeess deseribed. However, llle process as described will result in a eore of an organic solvent, an oily pl-ase or a particulate substanee. This will 5 limit the na~ure of ~he aclive agent ~hal can be eneapsulate(l. For example, many of Ihe organic solvents or solvent systems described would affeet Ihe ~stability of biologieally active agents, sueh as peptides and proleins, and would be likely to Iead to denaturation thereof and loss of pharmacological ac~ivily. l lle document does not deseribe the 10 iormation Or aqueous-rllled middle size nanoeapsules nor small diameter nanoeap~sules.
Olllel examples ol` microcap~sular drug carrier systems inelude lipo~some~s wllie}l are ~small pllo~spl~olipid ba~sed vesieles having an aqueou~s core. Lipo~somes obtailled by crosslinking of leeithin have 15 lipoidic wall~s ~struelurally related to those of biological membranes and as sueh have a ~shell defined by a molecular bi-layer.
Using leeilhin i~ is possible to syn~hesize small diameter vesieles (20-50 nm). However, liposomes are difficult to manufacture on an industrial scale. It is found that entrapped agents desorb very rapidly 20 from liposomes into the bloodstream, such that drug delivery to pha~oeytic cells i~s not achieved.
This desorp~ion of active agent and hence the lirnited stability of liposome~s hs due principally to the rapid hydrolysis thereof by blood enzyme~s.
Moreover~ the enLrapment levels of aclive agent achieved with liposome.s are low (see ~I Khoul-i ~allouh. N. (1986) sllpr-a).
Way.s of implovillg ~rug delivery, so as to aehieve better bioavailability and pharlllacokinetics are constantly being sought, e~specially for active agent~s which are subjeeted to rapid degradation following aclminkstratioll.
~ 94/15590 PC'rlIE94/00001 2t533~3 Oral adminis~ralion is one of ~he modes of administering drugs wl-ich has the grea~est clegree of patient compliance and thus ways are constantly being sougl-t Or formulating active agents for administration by the oral route whicl- it has not hitherto been possible to administer S by that route.
'~he low hydrolytic stability of liposomes means that they cannot be used for lhe delivery Or active agents by Lhe oral route. Enzymes of ~he gastrointeslinal lrac~ rapidly destroy liposomes following administration ~ tlle oral route with release of active agent, so that 10 uptake of active agent fronl the intestinal tract into the bloodstream is nol reali~.e~.
Di~clo~sure of Invention The invenlion provides nanocapsules comprising a polymeric shell formed of a surface active poly(alkyl cyanoacrylate) material 15 arranged in one or more layers.
The nanocapsules according to the invention are stable and can be used to entrap effective amounts of an active agent. lt is possible to achieve a degree Or encapsulation of 75% or higher with the nanocapsules according to the invention.
E3y polymer herein as regards the polymeric shell is meant any suitable polymer according to the l.U.P.A.C. definition of polymer.
The polymeric shell of the nanocapsules according to the invention is made up of polymer chains typically of the order of 10 or more monomer units. The polymer shell formed has the ordered arrangement of a mono-, bi-, ~ri-, or polymolecular layer typical of a liposorne.
Preferably, the nanocapsules in accordance with the invention have a ~iameter in tl-e range 20-150 nm.
-~ 94/15590 PCTIIE94/00001 21533~3 l he size of the nallocapsules is determined by the type of monomer used and by ttle melhod of preparation as hereinafter described.
l`he presence of one or more ordered layer(s) in the polymeric 5 shell follows from lhe size of the nanocapsules produced and may be confirmed by electron diffractioll.
The nanocapsules according to the invention are primarily intended for use in tlle delivery of active agents to the human or animal body, includillg delivery for the purposes of medical diagnosis 10 involving imagil~g. However, lhe nanocapsules according to the inventioll are not limiled to such use and will also find application in agriculture, in cosmetics for delivery of a wide variety of active agents including fragrances, ~hc food industry and other areas of technology to which their properties are adapted to provide a desired effect. For 15 example, the nanocapsules according to the invention are ideally suited for the encapsulation and subsequent delivery of systemic fungicides, herbicides and pestici(les alld plant growth controlling agents to plants.
Thus, typically an active agent is contained in the aqueous- or non-aqueous phase contained in the core.
An especially preferred polymeric material for the nanocapsular shell is a poly(alkyl cyanoacrylate) material, more especially a surface active poly(alkyl 2-cyanoacrylate) having the general formula:
/CN
COOR "
~ 94/15590 PCT/IE94/00001 6 21~3393 wherein R i s --CH2 t CH2~mC~ 3 --CH2 ~ CH2~m COOH, ~ CH2 ~CH2tmcOoR, or--CH2CH2--0~ CH2CH2 ~ ~m R;
R' is--CH3,--CH~ ~CH2~l,CH3.--C~ CH33~ CH2-C(CH3)3~ or --O--Ar;
m has a value Or from () to 20; and n has a value of from I to 20 According to a preferred melhod, the nanocapsules are formed 5 by in~erfacial polymerisation of self-arranged micelles of surface active cyanoacrylate monomcrs in an aqueous phase as hereinafter described The active agent encapsulated in ~he nanocapsules according to the invention is any water soluble or water insoluble active agent, including naturally occurring substances and synthetic analogues 1 0 thereof Given that the active agent is dissolved or dispersed in an aqueous or non-aqueous phase in the core of the nanocapsule, the stability thereof is maximised Especially preferred active agents for encapsulation in the 15 nanocapsules according lo the invention are water soluble active agents Such preferred active agents include arnino acids, peptides and polypepti~les Such aclive agents include hormones, hormone release factors, cytokines, encephalins, blood factors and products including enzymes and anlibodies, and other active agents which are susceptible 20 to degradation and/or modification by proteolytic and other enzymes before exerting lheir effect, especially if administered by the oral route. 'I he latter type of active agents also includes anti-tumour agents, antibiotics~ opiates ~such as apomorphine, dopamine, serotonin and ` ~94/15590 PCTIE:94100001 21~33~3 other agents active on tlle cenlral nervous system, and steroid hormones such as proge~sterorle and te~slosterone.
When the aclive agent is an antibody, the antibody can be a monoclonal or polyclonal anlibody.
'I he nanocapsules according to the invention are also especially suitable for the encapsulation and subsequent delivery of immunomodulating agen~s, for example, cyclosporin.
l`he nanoc.lpsulcs according to the invention can also be used to encapsulate variou.s VaCCillC~s.
lt will be appl-cciatcd that the nanocapsules according to the invention can inclea~se lhe bioavailability and efficacy of a wide range of water soluble ac(ive agents by protecting said agents from premature degradation in the gaslrointestinal tract and Lhe blood and allowing for a sustained release thereof~
The invention also provides an active agent delivery system comprising nanocapsules as hereinbefore described.
The nanocapsules according to the invention are stable and release their contents on degradation following ~(lministration to the target system or locus.
The nanocapsules according to the invention when intended to deliver an active agent for use in therapy or prophylaxis may be administered orally, parenterally or topically to the human or animal body. Following oral administration the nanocapsules traverse the gut wall and are taken up into the blood stream and the product is released on degradation of lhe nanocapsule shell or wall.
The nanocapsules are useful in delivering active agents to the blood stream by ~he oral rou~e lhat are not normally suitable for administration by ~his rou~e in traditional conventional pharmaceutical ` ~94/15590 PCT/IE94/00001 21~33~3 fortnulations. I he cncapsulated product is protected from the harsh condilions of lhe gul, SUC}l that a significantly greater proportion of active agent is delivere(l ~o t}-e bloodstream than would be possible by simple oral administration of the non-encapsulated active agent. For 5 example, insulin, a prolein, is normally given by intramuscular injection. If given orally i~ is normally degraded by the normal diges~ive processes of ~l~e gut an(l only a very small and variable proportion finds i~s way in~o ~lle bloodstream. lnsulin encapsulated by the met~lod according ~o ~he invention can be given orally wi~h minimal 10 lo~s~s of pharmacological effec~ Thus, there are major benefits for the patien~ botll in ~CI lllS of reducing stress and increasing convenience Suitable formulatiolls of the nanocapsules according to the invention for adlllinistration by the oral route include capsules, dragées, elixins, granules, lozenges, pellets, powders, suspensions and 15 tablel~s. In the ca~se of tablets care should be taken that the tabletting ~echnique doe~s no~ Icad ~o any disruption of ~he nanocapsules and alteration of their release properties. Such tablets can be formulatecl for rapid disintegration in the gastric and/or intestinal juices, if required or, alternatively, coated so as to further delay the release of 20 the active agent The nanocapsules can also be formulated as solutions or suspensions for injection intramuscularly, intravenously and subcutaneously 1~ is also possible to formulate the nanocapsules accordillg to tl)c invention in liquid form for administration by 25 perfusion.
Further types of formulations according to the invention include nasal formulations, ocular agents, including slow release implants containing the nanocapsules, pessaries, suppositories, lozenges coated on one surl;.lce witll a bioadhesive for use in the buccal cavity or 30 formulations for adlllinis~erillg an active agent sublingually~
` ~4/15590 PCT/IE94/00001 21~33~3 'I`lle nanocap.sllle~s will gellerally be formulated in unit dosage lorlll for a~lminislr.llion or applicalion in an amount and for a time pre~scribe~ by all a(len~ing pllysician.
A preferre(l me~llotl for preparing lhe nanocapsules aeeording to 5 the invention comprise~s inlerracial polymerisation of a surfaee aetive cyanoacrylale mollolllel in the form of a colloidal solution eomposed Or self-arranged micelle~s in an aqueous medium under polymerisation inilialing condilioll.s. Colloidal parlicles of an aclive agent to be eneapsulaled may serve a~ the initia~or of polymerisa~ion. However, 10 polymeri.salion can also be spontaneou~s. The aqueous medium is preferably compo.sed ol` a lwo phase aqueou~s system.
A single or mollo-pllase aqueous system for use in aeeordanee with the invenlion i~s typically a solution physiologieally isotonic in s~reng~h and eomprises wa~er or an aqueous solution of one or more ]5 water-soluble polymer~s and/or one or more water soluble salts.
~ two pha~se aqueou~s system for u~se in aceordanee with the invention preferably comprises an aqueous eolloidal solution of two or more water soluble imtniseible polymers. Sueh water soluble immiseible polylllel-~s are known (see for example, Alberdsson, P.A.
20 "l'artition of eell par~ieles and maeromolecules", Wiley, lnternational Scientific N.Y. (1971) pp 30-37).
lhe polymers are selected primarily on the basis of their eompalibilily ancl den.sity. As regards the former criterion, there should be little or no affinity between the polymers, sueh that they do 25 not form aggregate~s or interaet unfavourably in solution.
'1 he followill~ are examples of suitable combinations of polymer~s:
DexLran sulphate an(l methylcellulose L~extran sulpl-ale and polyethyleneglyeol l. ~
` 1~94/1~590 PCT11E94100001 2I~3313 l)exlran sulpllale antl polyvinylalcohol Dielhylaminoelllyldextran and polyelhyleneglycol Dextran and polyvinylalcohol Dextlan and melhylcellulose Dextran and polyethyleneglycol Dextran and T~icoll (l~icoll is a Trade Mark) Dextran and oxypropyldextran L)extran and a mixed polymer of ethylene oxide and propylene glycol sucll as l'lurollic (I'luronic is a Trade Mark) Dextran alld polypropyleneglycol Oxypropyldextran and polyethyleneglycol Sodium carboxymelhyldextran and polyvinylpyrrolidone Dex~ran and chi~osan Dexlran and dextran sodium sulphate The formalion of a stable ~wo phase system also depends on the concentralions Or tllc rcspeclive polymers in the solution. lf the concentration of the polymers is below a critical level, then the two aqueous polymers will not separate into layers. The behaviour and characterislics Or difrerent polymers in combination must be 20 determined empirically (see Alberdsson, P.A. (1971) sl-pra).
The polymerisalion initiating conditions preferably involve the use of an initiator Or anionic polymerisation, which initiator is located within colloidal par~icles of a discrete phase or within a continuous phase. Examples of anionic polymerisation initiators include substances 25 containing nucleophilic groups such as, for example, amines and thiols.
Generally, the conlinuous phase will be present in a large excess relative to the discrcte phase, for example in a ratio of 100:1-50:1.
The interfacial micelles of cyanoacrylate monomer preferably sel~-arrange on the surface of colloidal particles of a discrete phase or 30 witllin solutioll. To provide a cyanoacrylate monomer having the ability lo self-arrangc in an aqueous medium to form micelles surface ` ~4/15590 PCT/E94100001 ~1S33~
active cyanoacrylic monomers of the type hereinabove defined are used.
In order to provi(le cyanoacrylate monomers having surface active propcrlies, hydrophilic or hydrophobic radicals are introduced 5 into the cyanoacrylate molecule as hereinafter described.
The synthesi~ of surface active cyanoacrylate monomers for use in accordance wiLh tlle invention is illustra~ed by the following preparation Example~s.
Suitablc aniollic typc surrace active alkyl 2-cyanoacrylates having 10 an ionogenic moiely in the ester radical have ~he general formula:
/CN
C~12=C o Il--O~CH2tnC~
O OH
wherein n has a value of from I to 20.
Suitable nonionic surface active alkyl 2-cyanoacrylates having a 15 hydrophilic moiety in the ester radical have one of the following formulae:
/CN
i) CH2=C ~0 C--O ~ CH2tn C
OR
` ~94tl5590 PCT/IE94/00001 12 21S334~
wherein R is--CH3,--CH2~CH2~mCH3~--CtCH3t2--CH2(CH3)3- or --O--Ar; an~
m and n each has a value of from I to 20;
CN
ii) CH2--C CH3 C~H3 C--O ~ CH2CH2O ~¢--CH2--I--CH3 wherein n has a value Or Irom I to 20; and /CN
iii) Cl~2=C
C--O ( CH2 )--CH3 wherein n has a value of from 0 lo 20.
The principal stages in the encapsulation process are as follows:
5 Stage 1: The preparation of a continuous phase.
Stage 2: Polymerisation of a cyanoacrylate monomer to form capsules.
.n~rlpsul;ltion using ;- mono-ph;l~e queous system ln Stage 1, an aqueous solution is formed for use as a polymerisation medium for obtaining capsules. Preferably, an 10 isoosmotic solution con~aining water soluble polymer and corresponding salts is used. An inhibitor of polymerisation, preferably cyanoacrylic acid, and a water soluble active substance are added to form a solution which is physiologically isotonic in strength.
` ~)4/15590 PCT/IE94/00001 In Stage 2. a surface active ester of cyanoacrylic acid is added wilh vigorous stirring, prcferably using sonication. Micelles of monomer are formed in a continuous phase and then the monomer polymerises to form a solid shell of polymer. To increase the rate of 5 polymerisa~ion an initiator having a basic nature sucll as hydroxyl ion or heating to a temperature of up to 60C is used. The active substance is encapsulated following polymerisation of the monomer to form solid micelles due to the difference in hydropl~ilicity inside and outside the micelles. Prererably the solid shell is composed of a mono- or bi-10 molecular laycr of polylllcr.
Encapsulation U.Sill~ a two pha.se sy.stem Encapsulation u~sing a colloidal .solution of a water insoluble ac~ivesubstance The preparation of the continuous phase in the Stage 1, is similar 15 to that for a mono phase aqueous system hereinabove described.
However, the active agent is added in the form of a solution in an organic solvent, preferably an alcohol, or in the form of a suitable aqueous solution containing one or more solubiliser(s) or other additives.
ln Stage 2, the size of colloid particles of the active agent is determined before addition of the monomer, and the colloid solution is sonicated if necessary. The surface active cyanoacrylate monomer is added with stirring an(l tlle micelles of monomer are formed on the surface of colloid particle~s of active substance. The solid shell is formed on the surface of colloid particles following polymerisation of the monomer ~o form a polymolecular layer of polymer. The surface of the colloid parlicles serves as the initiator of polymerisation while a bulk phase serve~s as a polymerisation inhibitor.
` ~94/1!;590 PCT/IE94/00001 21~33~3 Encapsulation using a two phase aqueous solution ln Stage 1, a two phase aqueous system is formed containing an aqueous solution of two or more immiscible water soluble polymers capable of forming such a lwo phase systeni as follows. The polymers 5 selectetl are dissolve~ in water, whereupon they settle into two layers, following equilibration of the respective polymers. This separation occurs primarily because the polymers are of different densities. If require(l, a further polymer which partitions selectively into one or other of the layers may be added during this stage, said further 10 polymer having ~he capabilily to selectively concentrate a target active agent ~o be a(l~ed in sta~e 2 and which it is desired to encapsulate in either one or the o~her phase of the two phase system.
The rate or time required for the sep~ration of the layers depends on the choice of the individual polymers. Left to gravity 15 alone, the separalion can take from several hours to several days.
Separation can be accelerated by centrifugation.
After separation, the two aqueous solutions, upper and lower, are decanted into separa~e flasks. At this point, two stable systems have been created in whicll the upper phase is equilibrated with lower phase 20 and l~ice l~er.~a. Both phases are used in the creation of an emulsion in Stage 2.
Stage 2:
In lhis stage~ an emulsion is formed between the two separated phases described above. Typically, the emulsion formed contains the 25 two phases in a ra~io of ~he order of 100:1. On emulsification, small droplets of the minor component form a discrete phase dispersed within a bulk or continuous phase of the major component which is present in excess. ~s both components in the emulsion have been mutually equilibrated in Stage l, the droplets are relatively stable in the 30 mixture and thus an emulsion can be formed.
~ 94/15~90 PCT/IE94100001 21~33~3 The emulsion is formed by vigorous agitation such as that achieved by means Or sonication or vortex mixing. The size of the droplets forming the discrete phase is primarily controlled by the degree and rate of agitation.
'I he active a~ent to be encapsula~ed is included in the emulsification proces.s. 'I he choice of upper or lower phase (created in Stage I ) to form the minor component in the emulsion is determined by ~he physical and chemical properlies of the active agent. Indeed, such properties also inlluence the choice of polymers used in Stage 1. The droplets within ~he emulsion become encapsulated by the alkyl 2-cyanoacrylate added in ~he next stage - Stage 3. Accordingly, it is desirable to selectively concentrate the active agent inside the droplets.
Thus the choice of upper or lower phase to form ~he droplets in the emulsion is primarily determined by the affinity of the active agent for IS the respective phases.
The method also allows for an initiator of polymerisation to be concentrated inside the droplets. In some instances, this may be the active agent itself. If this is not an initiator, however, this must also be added at this stage.
Stage 3:
In this stage, an alkyl 2-cyanoacrylate is added and the droplets are encapsulated following polymerisation at the droplet surface. Alkyl 2-cyanoacrylatcs polymerise on contact with an initiator of polymerisation. Nucleophilic chemical groups are good initiators of polymerisation. Conversely, acids, particularly strong acids, inhibit the polymerisation process. ln the emulsion created in Stage 2, polymerisation in the bulk phase or continuous phase is inhibited by a low pH. This effect is reversed when the monomers encounter the initiator at the surface of the droplet, resulting in encapsulation by polymerisation of the droplet which includes the active agent.
` 9~94/15590 PCTlIE94tOOOOl 16 21~33~
Apart from the degree of agitation a number of other factors delermine the size Or tlle nanocapsules according to the invention. ln general, the more rapid thc rate of polymerisation, the lesser the degree of control over polymerisation and the greater the size of the 5 nanocapsules formed.
Furthermore, the rale of polymerisation is inversely proportional lo the size of the alkyl group in the alkyl cyanoacrylate monomer, so that the larger the alkyl group, the slower the polymerisation and hence the smaller the nanocapsules formed.
Also the lower the pH of the aqueous solution, the slower the rate of polymerisation and lhe smaller the size of the nanocapsules formed.
The rate of polymerisation is primarily controlled by pH and the size of the ester (alkyl) group~s.
Best Mode~s for Carrying Out the Invention The invention will be further illustrated by the following Examples.
In the following Examples the method according to the invention is exemplified by lhe encapsulation of a dye (DiI) and the peptide type hormone oxytocin.
Example I
2-Cyanoacrylic acid e~sler of polyethylene glycol 4-tert-octylphenyl ether A 500 ml flask was fitted with mechanical stirrer, thermometer, argon and sulphur dioxide inlet adaptors, dosing funnel protected with 25 a drying tube, and Liebig condenser arranged for distillation. The flask was charged with 250 ml of anhydrous toluene, and 1 g of 2-cyanoacrylic acid was added to the boiling solvent with stirring and ~ ~94/15590 PCTtIE94/00001 21S33~3 sparging wi~h argoln 2U ml Or toluene/waler azeotrope was removed by dislillation antl 2.2 g Or phosphorus penlachloride dissolved in S0 ml of dry benzel~e was then added dropwise wiLh stirring and constant removal of benzelle by di~slillaliol~ e mixture was stirred under reflux for I llour and Lhen sparged with sulphur dioxide, while 100 ml of toluene containing by-product phosphorus oxychloride was distilled Orr to leave a resi(lual colourless solu~ion Or 2-cyanoacryloyl chloride in toluene. ~ solu~ion of 7.4 g of Triton-X100 (Triton is a Tradle Mark) and 0.5 g of hydroquinone in 50 ml of benzene was then added clropwi.se to l}le 2-cyalloacryloyl chloride solution with stirring and constant removal Or benzene by distillatioll. l he mixture was refluxed during I llour, coolcd, an(l solvent was dislilled off in vacuum to give 8.1 g of a colourless oil which was washed with hot hexane to give 7.8 g of tl-e 2-cyanoacrylale ester of polyethylene glycol 4-tert-octylphenyl elher. Elemen~al Analysis Calculated for C3gH63NO2: C
67.35%, H 9.30%, N 2.07%, Found C 66.7%, H 9.6%, N 1.9%, lH
NMR in 1: I C6D(,: (CD ~)2CO 0.75 (9H, s, (CH3)3C-~, 1.37 (6H, s, (CH3)2C-), 1.77 (2H, s, CH2), 3.62 (m, CH2O-), 3.98 (t, J = 4 Hz, CH2O-), ~.13 (t, J = 5 ~Iz, CH2O-), 4.43 (m, 2H, CH2OCO-), 6.08 (s, 1H, ~-C=C-), 6.67 (~. I H, H-C=C-), 6.88 and 7.33 (2d, each 2H, A2B2, J = 7.2 Hz, aryl) ppm.
Example 2 2'-Carboxyethyl 2-cyanoacrylate 9.~ g of 2-cyanoacrylic acid, 0.2 g of 4-toluenesulphonic acid and 0.1 g of hydroquinone were dissolved in 250 ml of anhydrous benzene in a S00 ml flask which had previously been washed with 10 %
sulphuric acid and dried USillg acetone, and which was fitted with a stirrer, a lhermometer~ sulphur dioxide and argon inlet adaptors, a dosing funnel and a Liebig condenser arranged for distillation. The solution was sparged wi~h sulphur dioxide and brought to reflux when a suspension of 9.9 g Or 3-hydroxypropionic acid in 200 ml of benzene was added dropwise wilh continuous removal of benzene-water azeotrope by disLillalioll. rhe mixture was heated with stirring and ~ 94/1559û PCTIIE94/00001 21~3q~
sparging with sulphur dioxide unlil the benzene-water azeotrope ceased to appear, and was then refluxed for a furlher 30 minutes. The volume was reduced to 100 ml by removal of solvent by distillation. The residual colourless solution was cooled~ filtered, and diluted with 500 5 ml of heptane to give 8.5 g of a solid which was collected. The solid was recrystallised from 1: I benzene: heptane which had been satura~ed wi~h sulphur dioxide lo yield 6.51 g of 2'-carboxyethyl 2-cyanoacrylate.
Example 3 Preparatioll of hexadecyl 2-cyanoacrylate lnlo a 0.5 litre flask fitted with mechanical stirrer, thermometer, argon inlet adaplor wilh a device for admitting a stream of gas under lhe surface of tlle reaclion mixture, a dosing funnel protected with a Drierite drying tube, a Liebig condenser provided with a vacuum distillation adaptor and a receiver flask connected to a vacuum flask was chargetl 0.98 g (0.01 mole) 2-cyanoacrylic acid, S0 mg methylhydroquinone, 200 ml dry benzene and 100 ml dry toluene. A
solution of 2.08 g (0.01 mole) phosphorus pentachloride in 50 ml of dry toluene was charged into the dosing funnel. While sparging with dry argon and stirring under reflux the phosphorus pentachloride solution was added dropwise. Following completion of the addition the reac~ion mixture was boiled for 15 minutes following which the reflux condenser was substituted by a Liebig condenser with a receiver and a calcium chloride drying tube and 200 ml of solvent were distilled off.
At this point 2.42 g (0.01 mole) n-hexadecyl alcohol in 50 rnl dry benzene was added from the dosing funnel while refluxing and stirring and sparging with dry argon. ~ollowing addition of the alcohol the mixture was boiled for one hour and then the solvent was distilled off to give 50 ml ren-aining which was cooled to 5C and left overnight (17 hours), following which crystals of 2-cyanoacrylic acid had fallen out WhiCIl were fillered off. The volatiles were removed by distillation under vacuum and the remaining solid recrystallised from hexane to give 1.57 g n-hexadecyl 2-cyanoacrylate (49% yield) solid; m.p. Sl-` \~94/15590 PCT/IE94/00001 ,9 21~33q3 3~C [Elemental Analysis Calculated for C2()H35NO2 C = 74.8, H =
10.9, N = 4.4, Found C = 73.5, H = 11.1, N = 4.1].
lH NMR ~6.24 (IH, s, CH~=C-), 5.38 (IH, s, CHb=C-), 3.89 (2H, t, J=5.8Hz, -CH2OCO-), 1.32 (281-1, m, (CH2)l4-), 0.91 (3H, t, -CH3) ppm. 13C NMR C ~13.62 CH~. 22.29 CH~CH2~ 31.56 CH3CH2CH2, 29.0(CH2)1(), 25.27 CH~ (CH2)12 CH2, 27.95 (CH~,(CH2)1~CH2), 65.98 CH3 (CH2)14 CH2O, 113.85 C, 115.99 CN, 159.78 C=O.
Example ~
l~nCapSUIalion itl a mono-phase system Preparation of.self-arranged poly(2'-carboxyethyl 2-cyanoacrylate) nanocap.sulc~s in a mono-phase aqueous medium.
A coolecl sonicalion reaction vessel was filled with 50 ml of a solution of 50 mg of citric acid (citric acid monohydrate obtained from Belgorodsky Plant of Citric Acid, Belgorod, Russia) in isoosmotic I S dextran-based plasma ~ubs~itule "Polyglukin" (Polyglukin is a Trade Mark of Krasnovarsky 1'1ant of Medical Preparation). The mixture was cooled and titraled by H3PO4 to a pH of 2.5-3.2. Approximately 0.3 g of 2'-carboxyethyl 2-cyanoacrylate (prepared in Exarnple 2) was added in portions wil}l continuous sonication and cooling of the reaction vessel to provide a reaction temperature not higher than 30C.
When the solution became cloudy adding of 2'-carboxyethyl 2-cyanoacrylale was sloppcd and the mixture was sonicated for 30 min.
with continuous cooling. The sonication should be stopped and the mixture cooled in the case of spontaneous heating. The nanocapsules obtained were sized by a Coulter Counter. ln the remaining Examples, the nanocapsules produccd were sized in the same way. The mean diameler Or nanocap~slllc.s obtained wa~s 25 nm. 95% of nanocapsule~
had a size of 25 nn~ .s~nLlard deviation 20 nm.
~94115590 PCT/IE94/00001 21~33~3 Example 5 Encapsulation of oxytocin in self-arran~ed nanocapsules f~rmed from the 2-cyanoacrvlic acid ester of polyethylene glYcol 4-tert-octylphenyl e~her using a mono-phase system A cooled sonicalion reaction vessel was filled with 50 ml of a solution of 50 mg of cilric acid in an isoosmotic salt-based solution.
The mixture was cooled an(l titrated wi~h phosphoric acid to a pH of 2.5-3.2. 2500 U of oxytocin was dissolved with stirring using a magnelic stirrer an(l 0.5 g of surface active 2-cyanoacrylic acid ester of polyethylelle glycol ~-tcrl-octylphenyl e~her monomer prepared in Example I was a~de(l dlopwise with conlinuous sonication and cooling of the reaction vessel to provide a reaction temperature not higher than 30"C. When the solution became cloudy adding of monomer was stopped and the mixture was sonicated for 15 min. with continuous cooling. The sonication should be stopped and the mixture cooled in the case of overheating. After this time the suspension is transferred to a magnetic stirrer and stirred for 24 hours. The pH is then adjusted to 7.2-7.4 by the addition of IN NaOH with continuous stirring. The nanocapsules produced were sized. The mean diameter of nanocapsules obtained was 48 nm. 95% of nanocapsules had a size of 48-49 nm, standard deviation 25 nm.
~xample 6 Encapsula~ion Or Dil in self-arranged nanocapsules formed from poly(hexadecvl 2-cyanoacrylate) In this Example. the method according to the invention is used to encapsulate the fluorescen~ dye named DiI (supplied by Molecular Probes Inc.. Eugene. Oregon, U.S.A.) useful for staining living cells in cell biology. Dil is insoluble in water and is a lipophylic substance accessible in tlle form of a solution in alcohol. Dil can be encapsulated 30 by the use of a s~rong lipophilic cyanoacrylate monomer in a mono-phase aqueous-organic medium.
~ 94/15590 PCT/IE94/00001 21~3313 A cooled .sonicalion reaclion vessel was rllled Wil}1 25 ml of citric buffer ~solution at pH=7.2-7.4 and 20 ml of alcohol containing I mg Dil. llle mixture was cooled with stirring using a magnetic stirrer and after complete di~ssolution of Dil the presence of colloidal micelles was 5 determined by a Coulter Coun~er. Further alcohol was added if necessary. I ml of alcol1ol solution containing 0.1 g of hexadecyl 2-cyanoacryla~e prepared in Example 3 was added dropwise with con~inuous sonica~ion alld cooling of Lhe reaction vessel to provide a re~ction temperalure not greatcr tllan 60C The suspension was 10 sonicaled for 30 mill~ willl continuous cooling. The sonication should be stopped and ~he mix~urc cooled in the case of overheating. Aher lhis time ~he suspen~sioll was transferred to a magnetic stirrer and stirred for 24 hours~ I he pH was then controlled and adjusted to 7.2-7.4 by lhe addition of I N NaOH with con~inuous stirring if necessary.
15 The nanocapsules produced were sized. The mean diameter of the nanocapsules oblained was 37 nm. 95% of the nanocapsules had a size of 37-38 nm standard deviation 25 nm. To estimate the yield of encapsulation the suspension was centrifuged at 45000 r.p.m. for 4 hour~ and ~he absorbance of the solution was determined by UV
20 spectro~scopy. Ihc es~ ated yield of encapsulation is 82%.
Example 7 Encapsulation of DiI in self-arranged nanocapsules formed from the 2-cyanoacrylic acid ester of polyethylene glycol 4-tert-octylphenyl ether In this Example a two phase aqueous system is used to 25 encapsula~e Dil. Under the appropriate conditions Dil is able to form an aqueous colloidal solulion.
A cooled sonicalion reaction vessel was filled with a 50 ml solution of 50 mg of citric acid in an isoosmotic salt-based solution.
The mixture was cooled and titrated with H3PO4 to a pH of 2.5-3.2.
30 mg of Dil in I ml of alcohol was added with sonication. The size of the micelles formed was determined by the use of a Coulter Counter and the mixture was sonica~ed until micelles with a size of 25-40 nm ` ~O4/15590 PCTIIE94/00001 22 21S33~3 were obtained. I llen 0.1 g Or the surface active 2-cyanoacrylic acid ester of polyelhylene glycol 4-tert-octylphenyl ether monomer prepared in Example I was added dropwise wi~h continuous sonication and cooling of the reaction vessel to provide a reaction temperature not greater than 30"C. The mixture was sonicated for 40 min. Sonication should be stopped and ~he mixture cooled in the case of overheating.
After tnis time the suspension was transferred to a magnetic stirrer and stirred for 24 hours. The pH was then adjusted to 7.2-7.4 by the addition of lN NaOH wilh continuous stirring. The nanocapsules produced were sized. The mean diameter of nanocapsules obtained was 115 nm. 95% of nanocapsules had a size of 115-116 nm, standard ~evia~ion 65 nm.
Example 8 Ençapsulation of Dil in self-arran~ed nanocapsules formed from ~he 2-cyanoacrylic acid es~çr of polyethylene glycol 4-tert-octylphenyl ether ln this Example, Dil was encapsulated using an aqueous colloidal solution of two immiscible aqueous soluble polymers exemplified by the use of a dextranlpolyethylene glycol (PEG) two phase system.
Preparation of the two phase system:
Dextran (10 g) and PEG (1.14 g) (supplied by Schuchardt, Munich, l~edcral Republic of Germany) were dissolved in 50 ml of water by mixing and heating to 80C. After cooling 50 mg of citric acid and 0.2 ml of H3PO4 were added. If required, further H3PO4 was 2S added to adjust tne pH to 2.5-3. The mixture was then allowed to stand and the layers formed separated in a separatory funnel.
Preparation of colloidal solution and polymerisation:
I mg Dil dissolved in I ml of alcohol was added dropwise to 1 ml of upper phasc (primarily PEG). The solution obtained was added ` ~94115590 PCT/IE94/00001 23 21~33~3 to 200 ml of lower phase (primarily dextran). The rnixture was then placed in a cooled sonication reaction vessel and was sonicated with continuous cooling to provide a droplet size of 60-80 nm. The size of droplets was determined by the use of a Coulter Counter as before.
5 Sonication was continued, if necessary. The surface active monomer 2-cyanoacrylic acid ester of polyethylene glycol 4-tert-octylphenyl ether obtained in Example I was added dropwise with continuous sonication and cooling of the reaction vessel to provide a reaction temperature not greater than 40~C. T}le mixture was sonicated for 40 rnin. Sonication 10 was stopped and lhe mixture cooled in the event of overheating. After this time t}1e suspellsion was transferred to a magnetic stirrer and stirred for 24 hours. The pH was then adjusted to 7.2-7.4 by the addition of IN NaOH wilh continuous stirring. The nanocapsules produced were sized. l he mean diameter of nanocapsules obtained was 135 nm. 95% Or tl-e nanocapsules having a size of 135 nm, standard deviation 55 nm.
Claims (17)
1. Nanocapsules comprising a polymeric shell formed of a surface active poly(alkyl cyanoacrylate) material arranged in one or more layers.
2. Nanocapsules according to Claim 1, having a diameter in the range 20-150 nm.
3. Nanocapsules according to Claim 1 or 2, wherein an aqueous phase is contained in the core defined by the polymeric shell.
4. Nanocapsules according to Claim 1 or 2, wherein a non-aqueous phase is contained in the core defined by the polymeric shell.
5. Nanocapsules according to any one of Claims 1-4, wherein an active agent is contained in the core.
6. Nanocapsules according to any preceding claim, wherein the poly(alkyl cyanoacrylate) material is a surface active poly(alkyl 2-cyanoacrylate) having the general formula:
wherein R is -CH2?CH2?mCH3,-CH2?CH2?mCOOH, -CH2?CH2?mCOOR, or -CH2CH2-O?CH2CH2-O?mR';
R' is -CH3,-CH2?CH2?mCH3,-C?CH3?2CH2-C(CH3)3, or -O-Ar;
m has a value of from 0 to 20; and n has a value of from 1 to 20.
wherein R is -CH2?CH2?mCH3,-CH2?CH2?mCOOH, -CH2?CH2?mCOOR, or -CH2CH2-O?CH2CH2-O?mR';
R' is -CH3,-CH2?CH2?mCH3,-C?CH3?2CH2-C(CH3)3, or -O-Ar;
m has a value of from 0 to 20; and n has a value of from 1 to 20.
7. Nanocapsules according to Claim 6, which are formed by the interfacial polymerisation of self-arranged micelles of cyanoacrylate monomers in an aqueous phase.
8. Nanocapsules according to Claim 6 or 7, which are formed by the interfacial polymerisation of a surface active cyanoacrylate monomer in the form of a colloidal solution composed of self-arranged micelles in an aqueous medium.
9. Nanocapsules according to Claim 7 or 8, wherein a mono-phase aqueous system is used.
10. Nanocapsules according to Claim 7 or 8, wherein a two phase system is used.
11. Nanocapsules according to any one of Claims 6-8 and 10, which are formed by interfacial polymerisation in a two phase aqueous polymeric emulsion.
12. Nanocapsules according to Claim 11, wherein the respective phases are present as a discrete phase and a continuous phase
13. Nanocapsules according to Claim 11 or 12, wherein the uptake of active agent in droplets of the discrete phase is promoted by adding one or more substances which cause the active agent to be expelled by the continuous phase.
14. Nanocapsules according to Claim 11 or 12, wherein the uptake of active agent in droplets of the discrete phase is promoted by adding one or more substances which cause the active agent to be attracted by said discrete phase.
15. Nanocapsules according to Claim 13 or 14, wherein the or each substance is a charged polymer.
16. Nanocapsules according to any one of Claims 5-15, wherein the active agent is a peptide or polypeptide.
17. An active agent delivery system comprising nanocapsules according to any one of Claims 1-16.
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| RU93001196 | 1993-01-11 | ||
| RU93001196 | 1993-01-11 | ||
| PCT/IE1994/000001 WO1994015590A1 (en) | 1993-01-11 | 1994-01-10 | Small diameter nanocapsules, process for their preparation and applications thereof |
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| WO1997049387A1 (en) * | 1996-06-27 | 1997-12-31 | G.D. Searle And Co. | Particles comprising amphiphilic copolymers, having a cross-linked shell domain and an interior core domain, useful for pharmaceutical and other applications |
| WO1998056361A1 (en) * | 1997-06-13 | 1998-12-17 | Medinova Medical Consulting Gmbh | Drug targeting system, method of its preparation and its use |
| ATE427743T1 (en) | 1999-11-15 | 2009-04-15 | Biocure Inc | POLYMERIC HOLLOW PARTICLES RESPONSIVE TO EXTRAORDINARY CONDITIONS |
| FR2806005B1 (en) * | 2000-03-10 | 2002-06-14 | Univ Claude Bernard Lyon | PROCESS FOR THE PREPARATION OF COLLOIDAL PARTICLES IN THE FORM OF NANOCAPSULES |
| EP1272231A1 (en) | 2000-04-06 | 2003-01-08 | University Technology Corporation | Compositions and methods for promoting wound healing |
| DE10111508A1 (en) * | 2001-03-09 | 2002-09-26 | Henkel Kgaa | Modified cyanoacrylate esters, nano- or microcapsules made from them and their use in detergents or cleaning agents |
| FR2839517A1 (en) * | 2002-05-07 | 2003-11-14 | Centre Nat Rech Scient | NOVEL BIODEGRADABLE SURFACTANTS OF THE AMPHIPHILIC COPOLYMER TYPE COMPRISING HYDROPHOBIC SEGMENTS AND OLIGO AND / OR POLYSACCHARIDES |
| JP2007525442A (en) * | 2003-03-05 | 2007-09-06 | ピーアール ファーマシューティカルズ，インコーポレイテッド | Oxytocin controlled release formulation and method of using oxytocin controlled release formulation |
| US7659423B1 (en) | 2006-04-18 | 2010-02-09 | Loctite (R&D) Limited | Method of preparing electron deficient olefins in polar solvents |
| US7569719B1 (en) | 2006-10-25 | 2009-08-04 | Loctite (R&D) Limited | Method of preparing electron deficient olefins |
| US7718821B1 (en) | 2006-12-19 | 2010-05-18 | Loctite (R&D) Limited | Method of preparing electron deficient olefins |
| US8053589B1 (en) | 2007-10-24 | 2011-11-08 | Henkel Ireland Limited | Imines and methods of preparing electron deficient olefins using such novel imines |
| CN101896454B (en) | 2007-10-24 | 2013-07-10 | 洛克泰特(R&D)有限公司 | Activated methylene reagents and curable compositions prepared therefrom |
| US7973119B1 (en) | 2007-10-24 | 2011-07-05 | Loctite (R&D) Limited | Adhesive systems using imines and salts thereof and precursurs to electron deficient olefins |
| CA2703603A1 (en) | 2007-10-24 | 2009-04-30 | Loctite (R&D) Limited | Electron deficient olefins |
| US8399698B1 (en) | 2008-10-24 | 2013-03-19 | Henkel Ireland Limited | Substituted activated methylene reagents and methods of using such reagents to form electron deficient olefins |
| US10196471B1 (en) | 2008-10-24 | 2019-02-05 | Henkel IP & Holding GmbH | Curable composition having an electron deficient olefin |
| TWI664224B (en) | 2014-12-12 | 2019-07-01 | 日商東亞合成股份有限公司 | Two-liquid type hardening composition |
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| FR2659554B1 (en) * | 1990-03-16 | 1994-09-30 | Oreal | COMPOSITION FOR THE COSMETIC AND / OR PHARMACEUTICAL TREATMENT OF THE TOP LAYERS OF THE EPIDERMIS BY TOPICAL APPLICATION TO THE SKIN AND PREPARATION METHOD THEREOF. |
-
1994
- 1994-01-10 AU AU57141/94A patent/AU5714194A/en not_active Abandoned
- 1994-01-10 PL PL31007294A patent/PL310072A1/en unknown
- 1994-01-10 WO PCT/IE1994/000001 patent/WO1994015590A1/en not_active Application Discontinuation
- 1994-01-10 EP EP94902998A patent/EP0682513A1/en not_active Withdrawn
- 1994-01-10 CA CA002153343A patent/CA2153343A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| WO1994015590A1 (en) | 1994-07-21 |
| AU5714194A (en) | 1994-08-15 |
| EP0682513A1 (en) | 1995-11-22 |
| PL310072A1 (en) | 1995-11-27 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |