Biomedical application of functional polymers

Functional groups, properly located on a poly-Subsequently, body enzymes ‘in situ’ cata- mer as well as its structure, are usually respon-lyze the release of the drug.. Biological molecu

Biomedical application of functional polymers

* Joseph Jagur-Grodzinski

The Weizmann Institute of Science, Rehovot 76100, Israel

Received 11 May 1998; accepted 27 May 1998

Keywords: Biomedical application; Functional polymers; Drug delivery

reduced The use of polymers in the The application of polymeric materials for stimulation and for immobilization of enzymesmedical purposes is growing very fast Polymers have recently been probed The application ofhave found applications in such diverse bio- synthetic polymers for gene therapy has alsomedical fields as tissue engineering, implanta- been investigated They may provide a safertion of medical devices and artificial organs, way of gene delivery than use of viruses asprostheses, ophthalmology, dentistry, bone re- vectors Polymeric materials have also exten-pair, and many other medical fields Polymer- sively been used for biosensors, in testingbased delivery systems enable controlled slow devices, and for bio-regulation Polymeric ma-release of drugs into the body They also make terial suitable for a biomedical application mustpossible targeting of drugs into sites of inflam- be ‘biocompatible’, at least on its surface.mation or tumors Prodrugs with macromolecu- Strictly speaking many polymeric systems usedlar carriers have been also used for such pur- for implantation of medical devices into theposes The term ‘prodrug’ has been coined to body are considered to be ‘biocompatible’,describe a harmless molecule which undergoes though after implantation they become isolated

immuno-a reimmuno-action inside the body to releimmuno-ase the immuno-active from the tissues of the body by collagenousdrug Polymeric prodrugs are obtained by con- encapsulation They are, therefore, actually re-

with appropriate drugs Such macromolecular induce any harmful effects, thanks to conjugates accumulate positively in tumors, tion of the biofilm generated on the surface.because the permeability of cell membranes of Interactions with the body are thus hindered.tumor cells is higher than that of normal cells Functional groups, properly located on a poly-Subsequently, body enzymes ‘in situ’ cata- mer as well as its structure, are usually respon-lyze the release of the drug Peripheral toxicities sible for its biocompatibility and / or biodeg-

encapsula-radability and may impart on it either

therapeu-*E-mail: cpjagur@weizmann.weizmann.ac.il tic or / and toxic characteristics For example,

1381-5148 / 99 / $ – see front matter  1999 Elsevier Science B.V All rights reserved.

P I I : S 1 3 8 1 - 5 1 4 8 ( 9 8 ) 0 0 0 5 4 - 6

carboxylic groups induce therapeutic activity of polymers may be converted into water solublemany drugs Cell and protein binding reactions as a result of ionization, protonation, or hy-and growth may strongly be affected by func- drolysis of side chains Such conversion doestional groups of an implanted polymer En- not significantly affect molecular weight, butcapsulation of an implant is triggered by ad- may be responsible for bioerosion in topicalsorption of various proteins on its surface and applications Progress in the field of tissueadhesion of cells from the adjacent tissues The engineering depends on development of novelnature of such biofilm may be strongly in- biocompatible fully bioresorbable polymers andfluenced by the surface properties of the poly- introduction of processing techniques, whichmeric material Cell and protein binding re- will enable reproducible three-dimensional ar-actions and growth of the attached cells can be chitecture on the macro- and nanometer scaleeffectively manipulated by appropriate function- [2].

As indicated above, an implanted polymeric microbial origin are being produced from material may be considered to be ‘biocompat- ral sources by fermentation processes They areible’, if its insertion into the body does not non-toxic and truly biodegradable [3] Biodegra-provoke an adverse reaction A thrombus is dation is usually catalyzed by enzymes and itformed very fast when polymers contact blood may involve both hydrolysis and oxidation.cells Materials with non-thrombogenic blood- Aliphatic chains are more flexible than aromaticcompatible surfaces must, therefore, be used in ones and can more easily fit into active sites ofcontact with the blood stream Truly biocompat- enzymes Hence, they are usually easier toible polymers, used for medical purposes, biodegrade Crystallinity hinders polymer degra-should be able to recognize and cooperate in dation Irregularities in chain morphology pre-harmony with bio-assemblies and living cells vent crystallization and favor degradation

Biological molecules and synthetic ligands, design appropriately functionalized, trulydesigned to fit cell surface receptors, which are biocompatible and biodegradable polymeric sys-able to induce specific healing pathways should tems Various surface treatments leading to its

be immobilized on the surface to induce such functionalization have been described in theeffects To achieve these goals, appropriate literature Low toxicity glycol methacrylatefunctional groups should be incorporated into resin embedding has been used for the immuno-surfaces, which should resist non-specific ad- histochemical assessment of tissue response, bysorption of proteins [1] Biocompatible poly- identifying inflammatory cells generated as re-mers used in biomedical applications must often sponse to contact with biomaterials A recently

be also biodegradable, and harmful products proposed procedure, based on infiltration step atshould not be generated as a result of their 2 708C and curing phase at 2 208C enables

biodegradation Biodegradable polymers (also processing tissues with implants in situ, withcalled bioerodible or bioresorbable) may be of retention of both the immunoreactivity andsynthetic or natural origin Non-toxic alcohols, biochemical potency of proteins and good pre-acids and other low molecular products, easily servation of morphology [4]

eliminated by the body, are formed as a result of Biological fouling of surfaces, due to hydrolysis ‘in vivo’ of such biocompatible sorption of proteins and / or adhesion of cells,polymers High molecular weight polymers with which may be highly undesirable in some cases,hydrolytically unstable crosslinks may be may be prevented by grafting onto surface suchbioeroded as a result of the release of their polymers as poly(N-isopropyl acrylamide) and

ad-crosslinked chains Finally, water insoluble its derivatives, or by deposition of a thin layer

Results of some of the recently conducted release may be triggered by cell enzymes or byinvestigations of these subjects are discussed in enzymes in the cell vicinity Local delivery may

inser-tion of biodegradable implants in the vicinity oflocations to be treated, or by introduction ofcatheters containing capsules either filled or

2 Drug delivery

coated with the slowly released drugs The release of drugs, absorbed or encapsu- degradable implants and nanoparticles withlated by polymers, involves their slow and appropriate drugs dispersed evenly in the poly-controllable diffusion from / or through polymer- meric matrix (monolithic dispersion) as well as

Bio-ic materials It represents a well established nanoparticles with a therapeutic agent adsorbedapproach to drug delivery Production of the at the surface or loaded into the core, have beenslow release (SR) drugs by the pharmaceutical formulated for such applications

industry is nowadays a matter of routine Drugs,

covalently attached to biodegradable polymers 2.1. Biodegradable and biocompatible

or dispersed in the polymeric matrix of such nanoparticles

macromolecules, may be released by erosion /

degradation of the polymer Both mechanisms Polymeric micelles often self-assemble whenmay sustain the release of therapeutic agent by block copolymers are used for their preparation.some systems Therapeutic molecules, com- Micelles, based on the biocompatible copoly-plexed by polymers, may be released from gels mers of poly(ethylene oxide (PEO) with poly(L-

by diffusion Release of the basic growth factor lactic acid) (PLA) or with poly(b-benzyl-Lmay be cited as an example of such system In partate) (PBLA), have been described in theothers, the release mechanism may simply literature [8,9] Synthesis of such nanospheresinvolve desorption of the adsorbed active agent with functional groups on their surface is sum-Recent research efforts have been concentrated marized in Fig 1

-as-on systems capable to target drugs towards sick Aldehyde groups on the surface of the PEO–organs or cells Systems, suitable for local PLA micelles may react with the lysine residuesdelivery in the vicinity of locations to be of cell’s proteins They may also be used fortreated, have also been investigated Such strate- attachment of the amino-containing ligands Thegies seem to provide the most effective ap- hydroxyl groups on the surface of the PEO–proach to drug therapy, and may represent one PBLA micelles can be further derivatized and

of the main trends of the future Targeting can conjugated with molecules capable to pilot the

be attained by attaching biomolecules, capable modified micelles to specific sites of a living

to recognize specific cells, to the surface of organism Such nanospheres have been tested asnanoparticles containing a therapeutic agent In vehicles for delivery of anti-inflammatory andthe absence of such recognition sites on their anti tumor drugs [10,11]

surface, less specific targeting can be achieved 80–150 nm in diameter nanoparticles of the

by using relatively large particles or macro- biocompatible and biodegradable polyester molecules as vehicles of drug delivery Such polymer PLG [poly(lactide-co-glycolide)] (cf.particles or macromolecules accumulate pref- Fig 2) have been prepared by the nanoprecipi-erentially in tumor cells, which are more perme- tation method (they have been precipitated withable than healthy ones The term ‘prodrug’ has acetone from their oily colloidal nanodispersionbeen coined as designation of a therapeutic in water) Thus formed particles of PLG were

co-Fig 1 (a) Poly(ethylene oxide)-co-b-benzyl- L -aspartate (PEO–PBLA) and (b) poly(ethylene oxide)-co- L -lactide (PEO–PLLa) micelles with aldehyde groups on their surface.

that PEG coating of nanospheres providesprotection against interaction with the bloodcomponents, which induce removal of the

Fig 2 Poly(lactide-co-glycolide) (PLG).

foreign particles from the blood It prolongs,therefore, their circulation in the blood stream.coated with 5–10 nm thick layer of the poly- In consequence, thus coated nanospheres may(propylene oxide)–poly(ethylene oxide) (PPO– function as circulation depots of the adminis-PEO) block copolymer or with the tetrafunc- tered drugs [21,22] Obvious therapeutical bene-tional (PEO–PPO) -N-CH CH -N-(PPO–2 2 2 fits can be achieved by slowly releasing drugsPEO) [12,13] Such coats are bound to the core2 into plasma, and thus altering their concen-

of the nanospheres by the hydrophobic interac- tration profiles About 200 nm in diameter tions of the PPO chains, while PEO chains coated nanospheres, in which PEG is chemicallyprotrude into the surrounding medium and form bound to the core have been prepared, in the

PEG-a steric bPEG-arrier, which hinders the PEG-adsorption of presence of monomethoxy PEG, by ring certain plasma proteins onto the surface of such ing polymerization (with stannous octoate asparticles On the other hand, the PEO coat catalyst) of such monomers as ´-caprolactone,enhances adsorption of certain other plasma lactide, and / or glycolide [22] Ring openingcomponents In consequence, the PEO-coated polymerization of these monomers in the pres-nanospheres are not recognized by macrophages ence of such multi functional hydroxy acids as

open-as foreign bodies and are not attacked by them citric, music, or tartaric, to which several

Derivatives of the phosphazene polymers, (MPEG–NH ) have been attached, yields mul-2

proved to be also suitable for biomedical appli- copolymer in which NH2 terminated methoxycations [15–17] Long-circulating in the blood, PEG molecules have been attached to tartaric

nanoparticles of the poly(organo phosphazenes It has been demonstrated that morphology,containing amino acid, have been prepared degradation, and drug encapsulation behavior of

mer) has been deposited on their surface structure Studies of nanoparticles composed of[18,19] Chemical formulae of such polyphos- the diblocks of the poly(DL-lactide-co-glycolide)phazene derivatives are shown in Fig 3 with the methoxy terminated polyethylene gly-Arterial infusion of the PLG nanoparticles col (PLG–PEG) or of the branched multiblocksseems also to be a promising candidate for PLA–(PEG) , in which three methoxy termi-3treatment of restenosis, caused by anginoplasty nated PEG chains are attached through a citric

spheres made from PLA, PLG, or other bio- polyester blocks form the solid inner core Thedegradable polymers such as e.g poly(´-cap- anchored to them, on the surface, polyetheralrolactone) (PCL), may be used for the intraven- (PEG) chains form a corona The nanoparticles,

tially identical polymers The only difference PLLA–PEG and the PDLA–PEG stereoisomers,between the respective notations is that the are shaped as discs with PEG chains sticking

Fig 3 Polyphosphazenes for medical applications.

out from their surface Their hydrophobic / hy- above described approach 400–600 nm

applications in cancer therapy and in gene glycopyranose hydroxyls of the dextran unitstherapy Such nanospheres are prepared by targeting moieties can be attached [24]

dispersing the methylene chloride solution of Nanoparticle of the biodegradable acrylicthe copolymer in water and allowing the solvent polymer poly(methylidene malonate) (PMM

to evaporate By attaching biotin to its free 2.1.2), which has ester groups as side branches,hydroxyl groups and complexing it with avidin, may also be useful for drug targeting [25]

studies of such systems [23] revealed that the been prepared by anionic polymerization offlexibility and mobility of the thus attached PEG ethyl - ethoxy - carbonyl - methyleno - oxycarbonylchains is similar to that of the unattached acrylate in the presence of 1% dextran (pH 5.5

spheres, with the PEG–dextran conjugates (cf Na HPO , at 258C, 24 h) [26] Glycolic acid3 4Fig 5) attached to their surface, have been and ethyl alcohol are formed as a result of itsrecently investigated as another variant of the hydrolysis Under the experimental conditions

Fig 4 Multiblock (PEG) –(X) copolymers Amino terminated methoxy polyethylene glycol molecules attached to tartaric acid with PLan m

side chains.

they have not been found cytotoxic Anotherdegradation pathway may, however, lead toformation of small amounts of formaldehyde.Main drawback of the earlier investigatedalkyl acrylates is their considerable cytotoxicity[26] Hydrogels suitable for delivery of pharma-ceutically active proteins and peptides haverecently prepared by attaching HEMA to dex-tran trough an oligolactate spacer [27] It hasbeen assumed that biocompatibility of suchpolymers is good, since their degradation prod-ucts are lactate, dextran, and HEMA

Fig 5 PEG–dextran conjugates.

2.2 Local drug delivery

Implants from biodegradable polymers, capsulating appropriate drugs or integrated withthem, are usually used for internal local drugdelivery This strategy enables delivery of ahigh local level of a drug at low level ofsystemic exposure and toxicity Thus hemor-rhage complications of systemic delivery of anantithrombotic agent may be avoided by deliv-ering it locally [28–31] Biodegradable, control-

en-Fig 6 Poly(methylidene malonate) Ester groups as side chains

biological processes and do not leave any tissue of rats cause adverse effects [33] Theseresidual implant structures Previously discussed investigators concluded that PLG is suitable for

in the section on nanoparticles biodagradable brain implants Such implants, in the form of

co-phazenes), as well as PLA, PGA, PCL, poly- polymers, loaded with 2% of heparin and coated(orthoesters) (POE), poly(hydroxybutyrate), with PLA, effectively inhibit thrombosis andpoly(alkane anhydrides), oxidized cellulose, col- stenosis [28–31]

lagen, gelatin, and various synthetic hydrogels, NMR and EPR studies of their laminates withare used as biodegradable implants Gelatin and poly(FAD–SA) [where, SA stands for sebaciccollagen, which biodegrade into amino acids acid and FAD for fatty (erucic) acid dimer (cf.and are eliminated from the body within a few Fig 7)] revealed [34,35] that the 50% FAD/days, as well as poly(alkane anhydrides), which 50% SA 2 mm thick tablets are almost com-biodegrade into aliphatic diacids and are elimi- pletely absorbed few days after implantation

useful for short-term release PLG copolymers, heparin was released at a constant rate for ateliminated from the body within 6–12 months, least 20 days On the other hand, the sameare also useful for relatively short-time drug copolymer uncoated by PLA released the ab-release The rate of their degradation depends sorbed by it heparin for 4 days only The

on the manufacturing procedure and on the possibility of using fast eroding polyanhydridescomposition of the copolymer Device elimina- laminated with the slowly eroding poly(DL-lactiction has been found to be much faster for the acid) for manufacturing implants capable to

conducted on animal models revealed that, in recently been investigated [36–38] Gopferichspite of the fact that these copolymers are used an analogous FAD copolymer (cf Fig 8)classified as biocompatible, they irritate cor- poly[1, 3 - bis( p - carboxyphenoxy)propane - co -

inflammations Even more extensive inflamma- coated with PLA’s (Mw 5 1900 and 17 400).tory response has been found to be evoked by He found, that such implants may release onesuch supposedly biocompatible polymers as drug in two phases or two different drugs onepoly(hydroxybutyrate–valerate), POE, and PCL after another Poly(FAD–SA) matrices have[32] Another work on PLG did not reveal, been also tested on animal models as suitablehowever, that implants of PLG in the brain materials for slow release of the anti-malaria

Fig 7 Polyerucic-co-sebacic acid (FAD–SA).

Fig 8 Polyh1,3-bis( p-carboxyphenoxy)propane-co-SAj (PCCP–SA).

tumor drug, carboplatin, may be effective in course, lower than those formed as a result oftreating gliomas [40] Poly(´-caprolactone) the degradation of polyesters Moreover, their(PCL) has been tested as a vehicle for slow biodegradation may be induced by the action ofrelease of drugs at tumor reaction sites It was both esterase and protease, and bioactive mole-used as a surgical paste loaded with the anti- cules may be attached to side groups of thetumor-drug taxol [41] An 80:20 copolymer of a-amino acids The preparation of the polydep-

PCL with DL-lactide: [poly(CL–DLLA)] (see sipeptides by a new method of acylation of anFig 9) has also been found to be a suitable amino acid with a hydroxy acid, followed bymaterial for slow release devices In vitro cyclization and ring opening polymerization ofexperiments with this polyester revealed that up the product, have recently been discussed [46]

to 80% of the incorporated drug were released Sequential polydepsipeptides had previously

days It has been found in these experiments phosgen Thus formed N-carboxy anhydrides

that a relatively slow release, which was initial- (NCA) of amino acids have been subsequently

ly diffusion controlled, became much faster reacted with 2-nitrophenylsulphonyl chlorideafter 50 days when its constant rate of release (NPS-Cl) After purification of the product, itsbecame governed by the rate of erosion of the reaction with hydroxy acid yielded NPS-didep-polymer For a lower molecular weight co- sipeptide Its reaction with the NCA derivativepolymer (Mw 5 120 000), this transition was of another -amino acid yielded an end protectedmuch sharper After the first 40 days, during tridepsipeptide (cf Fig 11) This step could bewhich rates of release of the drug by the two repeated to form tetra-and pentadepsipeptides.copolymers were identical, the remaining drug Thus obtained end-protected oligodepsipep-

Biodegradable-biocompatible ceramers may hydrochloride and purified by recrystallization

cells They have been prepared by reacting treated with a small excess of triethylamine and

hydroxyls at both ends [43–45] Acid catalyzed polydepsipeptide [47] The sequential hydrolysis of the so formed bis(triethoxysilane) psipeptide AGL (cf Fig 12) has been tested asPCL, followed by crosslinking with Si(OH) ,4 a biodagradable matrix for drug release.yields a three dimensional network of a hybrid The LH-RH agonist was incorporated intoorganic–inorganic ceramer (cf Fig 10) Such AGL matrix Its release from such implants wasresorbable bioactive ceramers may be useful for probed for treatment of prostates (on animal

preferable to the previously described polyester after 24 weeks

type biocompatible-biodegradable polymers

2.3 Inert biocompatible polymers as implants

Implants with scaffolds made from polymericmaterials, which are not biodegradable, havealso been described in the literature: EVA(copolymer of polyethylene with poly(vinyl

Fig 9 Poly(caprolactone-co- -lactide) (P[CL–DLLa]). acetate), has been used for slow release of

Fig 10 Three dimensional network of a hybrid organic–inorganic ceramer.

growth-factors, antibodies, antigens, and other fluorocarbon layer, have also been used for the

pH-encapsulating Langerhans islets, has been tested sensitive (it dissolves in water at pH 5.5), has

as a component of bioartificial pancreas [49] been used for the encapsulation of drugs to be

‘In vivo’ and ‘in vitro’ tests suggested that such delivered in the first part of the intestine [50].membranes, obtained by casting into water 15%

solution of the polyamide in formic acid, have 2.4. Prodrugs with polymeric carriers

been biocompatible Permeability characteristics

of such membranes have been improved by It has been pointed out in the introduction toaddition of 1% of PVP to the casting solution this review, that conjugation of drugs with

membranes, coated to increase their hemocom- accumulation in cancerous cells Tethers atpatibility by plasma deposition of a smooth which drugs are attached to macromolecules

Fig 11 Synthesis of a sequential polydepsipeptide.

functional groups may be available in themacromolecules for bounding complexing moi-eties, responsible for targeted and site-selectivedrug delivery Antitumor drugs connected by a

peptide tether with the

poly[N-(2-hydroxy-propyl)methacrylamide] copolymer have beenextensively investigated Its slow releasingconjugate with the antitumor drug adriamycin

Fig 12 Sequential polydepsipeptide AGL

(alanine–alanine–N-(2-(attached through tripeptide spacer), have been

hydroxyethyl)- L -glutamine–lactic acid).

found to increase effectively animal life span[51] It reached the stage of clinical evaluation[52]

present in or near tumor cells, thus enabling ‘in through a variety oligopeptides with the solublesitu’ release of the active drug Moreover, polymeric carrier: poly[N-(2-hydroxyethyl)-L-

Fig 13 Mitomycin C attached through an oligopeptide spacer to polyhN-(2-hydroxyethyl)-L -glutamine j (water soluble carrier).

glutamine] (cf Fig 13), have been found to radioimmunotherapy and for diagnostic perform much better than the free drug Its poses, have been discussed

pur-systemic toxicity was lower, and activity against

animal models of established tumors much

higher than that of the unconjugated drug While 3 Bone bonding and repairing

free drug showed no activity against C26

the life span was achieved with its prodrug This phosphate crystals in a collagenous matrix

as spacer, reached the stage of clinical trials It take place when a layer of apatiteappears to be an excellent candidate for treat- [Ca (PO ) (OH)], similar to bone in crystallini-5 4 3

Controlled slow release of estrone hormone, on their surface in contact with blood plasma.through conjugation with starch, has recently Biocompatible materials which will providebeen investigated [55] Reaction of starch with matrix for ‘in vivo’ formation of hydroxyapatitebromoacetyl bromide provided active site for deposits may be suitable for bone repair.subsequent coupling of estrone as well as a The possibility of using a chemically modi-spacer between the drug and the carrier (cf Fig fied bamboo, a natural self-reinforced compo-

The prodrug approach, for site-selective de- solvent extraction of its cytotoxic componentslivery of various radiopharmaceuticals for and pretreatment with g-glycidyl-oxypropyl tri-

methoxypropane it was reacted with the end-capped PEO (cf Fig 15)

amino-In the thus treated material immersed incalcification solution a continuous layer of

Fig 14 Estrone conjugated with starch through acetyl group. CaHPO has been formed ‘in vitro’

Complex-Fig 15 Graft of the end-aminated PEG on bamboo.

ation of calcium ions by PEO chains may be sion of this gel-forming bioresorbable polymerexpected to provide strong bonds between the has been used in dental surgery to promoteinorganic crystals and the polymeric network osteoconduction After fulfilling its purpose itOrganically modified silicates (ormosils) con- was progressively depolimerized be the enzymetaining Ca(II) ions, which have been prepared lysozyme, and 6 months after surgery was not

by the sol-gel process [57], have been found to longer detectable in the body [59] Modified

be bioactive Apatite layer is deposited on them chitins have also been applied for dressings ofwhen they are immersed in a simulated body wounded soft tissues and controlled delivery offluid Such fracture tough and relatively strong anti-inflammatory drugs [60]

bioactive compounds seem to be promising A composite of the modified chitin fibers in amaterials for direct bonding to living bones PLLA matrix could be a potential candidate forChitosan and its derivatives (cf Fig 16) have use in bioadsorbable devices on load-bearingbeen found to be of help for repair of bone bones [61] The ultimate shear stress that thedefects and regeneration of bone tissues, thanks fiber-matrix interface could withstood seems to

Methylpyrrolidone chitosan, which randomly Self reinforced polyglycolide (PGA) carries pyrrolidone rings covalently attached to ites have also been proposed for bone repair andthe polysaccharide backbone was obtained by regeneration [31] Use of absorbable polymerschemical modification of chitosan [58] Emul- for such devices obviates the need of operation

compos-Fig 16 Chemical formulae of chitin and chitosans.

for implant removal, while eliminating risks due A socket made from ultra high molecular

to the permanent presence of foreign material in weight polyethylene (UHMWPE), Mw 5 2–5 3

6

the surface by g-radiation of slightly over 100Mrad, in combination with the zirconium or3.1 Non-absorbable polymers

alumina femoral head, seems to be the bestPoly(tetrafluoroethylene) (PTFE), thanks to material for total hip prostheses [64]

its inertness, could be applied without any Decrease in the thickness of such sockets, at

5

chemical modification as a prosthetic material steady state wear (after 10 3 10 cycles underNevertheless, it be desirable for many medical 250 kg load and saline water lubrication), was

6

devices to functionalize its surface It can thus less than 20 mm per 10 cycles and their swing

be made hydrophilic Its biocompatibility can be frictional torque was 3.34–9.02 Nm

altered and biological response to the implanted

material tailored Hydrophilized microporous

used as components of immunoisolation devices

for generation of antitumor immunity, through Certain chemical entities produced by indirect presentation of antigen [62] It has been teria, fungi, as well as of mammalian origin,shown recently [63], that very thin outside layer stimulate antibody production, rejection of

irradiation (lmax5 366 nm) (in nitrogen atmos- immune system Many of these substances havephere) Such treatment of samples, in contact saccharide, oligosaccharide, polysaccharide, orwith the DMF solution of benzophenone plus peptide fragments Some may be extracted fromsodium hydride, causes extensive defluorination natural sources, other may be activated by

of the PTFE surface, accompanied by intro- chemical modification [66] Glucanes with aduction of unsaturation and incorporation of liner backbone of b-(1,3)-linked D-glucopyran-oxygens Morphological damage induced by this osyl groups with various degrees of branchingprocess is smaller than that due to other treat- from the C6 position (cf Fig 17) have beenments The activated surface can be further found to be such effective, polysaccharide type,modified by various techniques of surface modi- immunostimulants

biologi-Fig 17 Chemical formula of glucane.

of the insoluble yeast glucan into a water

soluble sulfate or phosphate makes it much less 5.1 Molecular conjugate vectors

toxic (see p 7 in Ref [66])

Conjugate vectors with a polycation as aOligopeptides extracted from natural sources,

component, seem to be particularly adaptable

as well as synthetic oligopeptides, also show

for this purpose Their design involves bindingimmunostimulating activity The pentapeptide

DNA plasmid to a positively charged thymopentin corresponding to the Erg–Lys–

macro-molecule to which a ligand, promoting Asp–Val–Tyr sequence, and another thymus

attach-ment to and penetration into specific cells, ispeptide, thymulin, have found clinical use in the

covalently linked Such conjugates should betreatment of autoimmune diseases, such as

spherical in shape and small enough ( , 30rheumatoid arthritis

kDa) to allow exit from the bloodstream Bychoosing an appropriate ligand, such conjugatesmay, in principle, be targeted to any type of

5 Polymeric vehicles (vectors) for gene and

cells

oligonucleotides transfer

‘Ex vivo’ and ‘in vivo’ strategies have been 5.1.1 Polylysine

tion of the ‘ex vivo’ strategies is limited to cells highly branched synthetic polymer that can be manipulated extracorporally A ethylenimine (PEI) and the ‘starburst’ poly-vector, which can cross various systemic bar- amidoamine dendrimer, have been used as theriers as well as mediate gene expression in a positively charged macromolecular componentselected population of cells, must be used for of such conjugates [69–75] (cf schema in Fig.the ‘in vivo’ gene transfer [67,68] Both viral 18) Some details illustrating the formation ofand non-viral vectors have been tested as ve- the transferin-polylysine conjugate vector arehicles for gene therapy Though viral vehicles shown in Fig 19a,b)

poly-Fig 18 Complex of a DNA plasmid with a molecular-conjugate vector.

Fig 19 (a) Transferin prepared for coupling trough attachment of the (2-pyridyldithio)propionate, and polylysine prepared for coupling trough attachment of a thiol (b) Transferin-polylysine conjugate (c) Synthesis of a partially gluconylated polylysine.

Very substantial increase in transfer efficiency ficiency of the polylysine / DNA complexes mayhas been obtained by linking the associated with be greatly enhanced by blocking some of itscationic liposomes DNA with a polylysine to ´-amino groups Dissociation of the DNA com-

which a targeting ligand has been attached [72] plex inside the cell is facilitated by such

electrostatic forces, but also facilitates its entry genes to nucleus machinery is increased It has

compact structure [69] The transfection ef- polylysine with 43% (plus minus 4%) of its

Fig 19 (continued )

´-amino groups blocked by gluconoyl residues, tion of certain cells with only limited was much more efficient to transfect various ty [74]

N-2-(hydroxy-unsubstituted polylysine [73] Partial gluconyla- propyl)metacrylamide - co - tion of polylysine is schematically depicted in ethyl methacrylate chloride (HPM–TAMA)

Hydrophilic cationic block copolymers self- DNA have spherical shape

assemble with DNA into a complex protected

on its surface by hydrophilic shielding Such 5.2. PVP

complexes of polyethylene glycol-co-poly(L

poly(N-alkyl-4-vinyl-DNA have shown ‘in vitro’ efficient transfec- pyridinium bromides) with DNA plasmids, have

also been found to be efficient for tion of mammalian cells [75]

transforma-5.3 PEI

Polyethyleneimine has the potential to havethe highest charge density among organicmacromolecules, since every third atom of PEI

is an amino nitrogen that can be protonated (cf.Fig 21)

PEI retains a substantial buffering capacity at

Fig 20 Polyethylene glycol–poly- L -lysine block copolymer

Fig 21 Schematic representation of branching of a polyethyleneimine (PEI).

may be related to its efficiency in transfection 22b) may be formed by repeating steps A and BThe optimal PEI cation / anion balance for ‘in [78,79] Beginning with the 5th generation ofvitro’ transfection is only slightly on the cat- the PAMAM dendrimers their shape becomesionic side, which is advantageous for in ‘vivo’ spheroidal Maximum efficiency of mediation ofdelivery [76] Accordingly, PEI seems to be a transfection of cells was observed for the 6thpromising vector for gene therapy as well as a generation of the PAMAM dendrimers, (it has

˚

Much larger dendrimers cannot, apparently,penetrate into the cells It has been observed,5.4 Polyamidoamine (PAMAM) dendrimers

dramati-believed to be very promising vectors of oligo- cally as a result of partial degradation of the

The mechanism of complex formation between butanol or water [81] Heterodispersed

been found to be entirely based on charge several tens kDa, is formed as a result of the

Methods used in synthesis of polypeptides by solvolysis in such solvents It is believed thathave been successfully used for the preparation the observed increase in transfection ‘is princi-

of dendrimers, which requires very high conver- pally due to the increase in flexibility thatsions and efficient workup procedures for sepa- enables the fractured dendrimer to be compactration from reagents Spherical, starburst, poly- when complexed with DNA and swell when

prepared starting with ammonia as a core

fol-lowed by Michael addition of methyl acrylate 5.5. Lipidic vector systems

(step A) and subsequent reaction with a large

excess of the ethylenediamine (step B), see Fig Ionically charged liposomes formed by

Higher generations of the dendrimers (cf Fig have been investigated as gene therapy vehicles

Fig 22 (a) Synthesis of the triamine core of the polyamidoamine dendrimers (b) ‘Sturburst’ polyamidoamine (PAMAM) dendrimers.

[82] Lipids with polycationic head groups are es prepared from 0.5 mg of poly(L-lysine)

are more efficient and less toxic than mono- spheres ( , 100 nm in diameter), which have a

Transfecting efficiencies of the lipopolysines mixed with the

3b-[N[(N9,N0-dimethylamino)-may be increased by orders of magnitude by ethane]carbomoyl]–cholesterol (DC–chol) were

DNA bound to a polycation could be also prepared by initially adding an excess of theencapsulated in a negatively as well as in a lipid, are quite stable and highly active transfec-positively charged liposoms [82–85] Complex- tants Lipid mixture with anionic groups in

excess can be also used for the entrapment ofthe Plasmid DNA–polycation complexes in theliposomes They are very compact and purifica-tion step is not required for their preparation.Such liposome entrapped DNA complexes rep-resent a new class of non-viral efficient vectorsfor gene therapy

Fig 23 Lipopolylysine. Activation of complement by the polycation–

DNA vectors interferes with intravenous gene ance of insulin immobilized on a surface delivery [86] It can be minimized by adjusting lyzed poly(methyl methacrylate) film has beenthe polycation to DNA ratio (expressed in terms studied [88] It was found that amount of insulin

hydro-of charge ratio) For the polylysine–DNA com- that have to be immobilized in such systems, toplexes it can be minimized by modifying their regulate cell behavior, could be much smallersurface by coating with polyethylene glycol, and than the amount of free insulin required for

by reducing length of the polylysine chains [86] stimulation of the same behavior Moreover, the

maximal mitogenic effect of the immobilizedinsulin was greater than that of free insulin.Immobilization of the bioregulators on polymers

6 Regulation of gene expression

exhibiting reversible phase change in response

It has recently been demonstrated that syn- to changes in environmental factors

(stimuli-thetic polyamides containing N-methylimid- responsive) has also been contemplated Insulin

azole and N-methylpyrrole amino acids have an has been covalently bound to a affinity and specificity for DNA comparable to sive copolymer N-isopropylacrylamide-co-

thermorespon-the naturally occurring DNA-binding proteins acrylic acid (IPAm-co-AA), grafted onto [87] Such oligoamides with 8 pyrrole or imida- styrene film [89] IPAm-co-AA phase-separateszole segments separated by the residue of the from solution above 328C It was shown that

poly-g-aminobutyric acid and ended with the residue amounts of insulin immobilized in such systems

of b-alanine followed by the dimethylamino- could be 10 to 100 times smaller than freepropyl amide (cf Fig 24a) permeate into cells insulin producing the same effect As a result ofand can inhibit the transcription of specific temperature changes viable cells may be de-

Pyrrole–imidazole and pyrrole–pyrrole pairs use of the thermoresponsive IPAm-co-AA can bind any predetermined DNA sequence A actions leading to the formation of such systempairing of imidazole opposite pyrrole (Im / Py) are depicted in Fig 25

Re-targets a G–C base pair, Py / Im Re-targets C–G Bacillus Stearothermophilus, which produces

base pair Py / Py combination targets T–A and pollulanase enzyme, has been immobilized by

The generality of these pairing rules has been free-radical initiated polymerization of demonstrated on a variety of sequences of 5 to amide containing 7% of bis-acrylamide as the

poly-(acrylamide) is toxic to many microorganisms ithas been successfully used for immobilization

of various cells Specific enzyme production of

7 Polymeric matrices for growth and

B Stearothermophilus cells immobilized in the

immobilization of cells

20% poly(acrylamide) gel solidified in oil phaseSynthetic extracellular matrices may be used was found to be about 15-fold higher than thatfor culture of mammalian cells and for pro- of free cells It seems that this system may findduction of artificial tissues and organs Bio- application in the production of pollulanase.signal molecules, such as growth factors and Such bacterial polysaccharides as aliginate orcytokines, immobilized on such systems without gellan are also used as immobilization matrixsignificant loss in their activity, may help to for cells and enzymes [3]

regulate proliferation, movement, secretion, and Polymeric microporous membranes are oftendifferentiation of attached cells and enable their used for the immobilization of cells and en-growth in the absence of serum The perform- zymes in bioreactors Easy handling and large