synthetic polymer systems in drug delivery

Drug Development Strategy Review

2001 Ashley Publications Ltd ISSN 1462-2416 345

Ashley Publicationswww.ashley-pub.com

1. Background

2. Medical need and existing treatment

3. Current research goals

4. Scientific rationale

5. Competitive environment

6. Expert opinion

Synthetic polymer systems in drug deliveryCameron AlexanderBiomaterials and Drug Delivery Group, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michaels Building, White Swan Road, Portsmouth, PO1 2DT, UK

The development of synthetic polymers for applications in drug delivery isreviewed, with particular reference to polymers that can be activated torelease a medicinal agent in vivo or that can respond to changes in environ-ment to enhance the effectiveness of therapy. The mechanisms by which thesepolymers are designed to deliver drugs are highlighted, along with the chal-lenges facing synthetic chemists and pharmaceutical scientists in designingnew and more active therapeutic vehicles. Currently, synthetic materials withbiomimetic properties are attracting growing attention as possible new dos-age formulations and the potential applications of these increasingly sophisti-cated polymers in cell-specific drug targeting and in the emerging field ofgene therapy are also considered. Finally, the potential development issues fordelivery of therapeutics using active or smart polymers are discussed with ananalysis of the future trends in this rapidly expanding area of research.

Keywords: controlled release, drug targeting, gene therapy, polymer-drug conjugates, polymers, responsive/smart polymers, site-specific delivery

Expert Opin. Emerging Drugs (2001) 6(2):345-363

1. Background

The use of natural and synthetic polymers as drug delivery vehicles is now well-established and the importance of polymers in this area can be judged by an estimatethat sales of pharmaceutical products using drug-delivery systems will reach someUS$50 billion worldwide by 2002, or 20% of total pharmaceutical sales [1].

Polymers are used in therapy in a great variety of forms, for example as latex partic-ulates, in resin formulations and as microcapsules or liposomes for generic delivery.These delivery strategies essentially use polymers as a synthetic matrix that deliversthe therapeutic agent via diffusion-control or biodegradation. There is however agrowing realisation that polymeric systems can be adapted to carry out far more com-plex tasks than merely the passive release of a drug over an extended time-scale. Thisis partly because it is now much more widely appreciated that natural mechanisms ofdisease control are intrinsically feedback controlled and also because there are sophis-ticated methods emerging for the synthesis of polymers with very specific activeproperties. For example, consideration of the dynamic control of chemical processesin the body by messenger species is inspiring researchers to develop new classes ofpolymeric drug delivery systems that exhibit a form of biomimicry in that they areable to vary their properties in response to signals or changes in their environment.Synthetic bio-inspired polymers are also being developed for solution-based applica-tions where specific molecular recognition or bio-adhesive interactions are activelyengaged in vivo to target particular sites for therapy.

This review focuses primarily on these new categories of smart materials andconsiders the emerging methods that rely on synthetic polymers as an active andintegral part of the drug delivery and therapeutic process.

Synthetic polymer systems in drug delivery

346 Expert Opin. Emerging Drugs (2001) 6(2)

2. Medical need and existing treatment

The concept of controlled drug delivery has arisen from theneed to direct therapeutic agents to specified biological targetsat the optimum dose and over the correct time-scale. Thisgoal has yet to be achieved for many disease states, but never-theless, the adoption of controlled delivery devices, particu-larly those using polymers, is becoming more prevalent. Thesepolymers are principally being developed as agents for in situtreatments of disorders where conventional methods involvemultiple injections or courses of treatment, which are bothuneconomic and problematic in terms of patient compliance.Prime candidates for this type of polymer-mediated release aretreatments for diabetes enabling the controlled delivery ofinsulin, and in cancer therapies, where the margins betweenactive and toxic doses are especially narrow. However, thereare also a number of other areas where macromolecular-baseddelivery systems are attractive, involving new concepts inmedicine such as polymer prodrugs and gene therapy. In theselatter cases, rather sophisticated macromolecular constructsare being developed which are able to target specific cells withhighly active agents without releasing the drug systemically, orwhich can potentially deliver a copy of a gene to a defectivecell in order to express a necessary protein. Thus, whilst thereare many existing and well-established needs for controlleddrug delivery that are being addressed, the emergence of newtherapeutic regimes suggests that many more will become partof medical practice in the near future.

3. Current research goals

The most active areas of current drug delivery research arethose centred on improving the transport of therapeutics todisease sites, enhancing the specificity of biological targetingand controlling the processes by which drugs are released. It isnot surprising that polymer technologies are very attractive inall these fields, as conjugation or complexation of a therapeu-tic to a polymer can confer protection against degradationin vivo, or, by employing specific functionalities built into apolymer backbone, enhance both the time-scale and selectiv-ity of drug release. The key goals in each of these areas and theresearch strategies now being adopted are considered below inthe context of both scientific challenge and medical need.

4. Scientific rationale

4.1 Controlled deliveryPolymer systems are already much-utilised as vehicles for con-trolled delivery. There has been a large volume of work involv-ing the encapsulation or coating of therapeutic agents in apolymer shell or matrix to effect a more sustained release pro-file and this field has been extensively reviewed [2-5]. In addi-tion, the development of polymer hydrogels for drug deliveryhas now advanced to the extent that a large number of poten-tial applications are now under active practical investigation

[6-8]. Despite the success of these systems, there are still manyimprovements that are needed in terms of specific targeting ofdrugs in vivo. These are predominantly concerned with moreprecise control of release, including the use of specific envi-ronmental triggers and/or chemical processes to liberate drugsfrom a carrier polymer and the design of wholly new biomi-metic materials that target therapeutics using biological recog-nition or targeting motifs.

4.1.1 Polymer therapeuticsAn exciting addition to the array of methods for controlledrelease has been the development of polymer-drug conjugates(polymer therapeutics) wherein the drug is connected to a poly-mer carrier via a cleavable linkage [9,10]. The efficacy of thisapproach has been demonstrated by studying the transport ofthese conjugates into carcinoma cells [11], exploiting theincreased vessel permeability and poor lymphatic drainageexhibited by cancerous tissue. The abnormal behaviour of solidtumours, known as the enhanced permeation and retention(EPR) effect [12] has been a major focus in cancer therapy [13]but there is nevertheless a desire to increase the specificity of tar-geting and to ensure that once in the tumour locus, the poly-mer therapeutic breaks down efficiently to liberate the drug. Asa result, complex multi-component systems are now beingdeveloped which are designed to fulfil a number of stringentrequirements. Firstly, the polymer-drug conjugate must be solu-ble, stable in the bloodstream and capable of entering the lyso-somal region of the tumour cell where it must be readilydegraded to release the active therapeutic. It is also highly desir-able that the polymer therapeutic enters only certain specifictypes of cell and that the drug is released in an active formexactly when it reaches the target site.

One way of achieving the latter is by linking the drug to thepolymer by a group that is a substrate for a particular degrada-tion pathway in vivo, which additionally offers the chance totailor the release rate. The evaluation of this technique isunderway in a number of laboratories: for example, internali-sation into human hepatocarcinoma cells (HepG2) and ovar-ian carcinoma (OVCAR-3) cells has been studied using 2-hydroxypropylmethacrylamide (HPMA) polymer therapeu-tics containing N-acetylgalactosamine or monoclonal OVTL-16 antibodies as cell targeting moieties and adriamycin (doxo-rubicin) as both therapeutic (topoisomerase Type II inhibitor)and fluorescent label [14]. The drug in this case was linked tothe polymer backbones via di- or tetra-peptide spacers (Gly-Gly, Gly-Phe-Leu-Gly) and the conjugates were found notonly to target the tumour cells but were shown by confocalmicroscopy to be internalised via endocytosis to cell lyso-somes. Release of adriamycin and intercalation with DNAwas demonstrated by intense fluorescence in HepG2 cellnuclei [15] and biodistribution studies in mice indicated signif-icantly higher levels of polymer-drug conjugates in tumourtissues [16,17].

This approach potentially confers a significant additionalbenefit, in that a membrane component, P-glycoprotein,

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Expert Opin. Emerging Drugs (2001) 6(2) 347

which acts as an energy dependent efflux pump to removecytotoxins [18,19] and which is overexpressed in multi-drugresistant (MDR) cells, is less effective at removing polymer-drug conjugates on account of their size. HPMA-adriamycinpolymer therapeutics have also been shown to be stronglyretained in MDR cells and the intracellular concentrationswere significantly higher after the same incubation time thanthat of the free drug.

A number of these polymer therapeutics are now undergo-ing clinical trials [21], including the Adriamycin (doxoru-bicin) conjugates (1, PK1, FCE-28068) and (2, PK2, FCE-28069) shown in Figure 1.

Co-polymer (1) (N-(2-hydroxypropyl)methacrylamide(HPMA)-co-HPMA-Gly-Phe-Leu-Gly-doxorubicin) hasshown greatly reduced toxicity (approximately 10-fold) com-pared to free doxorubicin and evidence of activity has alsobeen obtained in chemotherapy patients at much lower doses.

A major advantage of the above polymers is the potential topromote further receptor-mediated targeting by the incorpo-ration of additional functional groups into the polymer back-bone and this, combined with the ability to adapt thepolymer-drug cleavage mechanism to specific enzyme action(polymer-directed enzyme prodrug therapy), suggests a prom-ising future use in cancer therapies.

4.1.2 Dendrimer drug delivery vehiclesThe development of hyper-branched polymers or dendrim-ers, specifically those of a monodisperse nature, has been amajor advance in polymer chemistry in the last fifteen years.

Whilst a full discussion of these polymers is beyond the scopeof this review, the close resemblance of some dendrimers tomicelles and even biological cellular structures has promptedan explosive growth in their development [21]. Dendrimersconsist of a core, from which polymer chains branch out inthree dimensions, forming a discrete macromolecular particleor micelle with a globular shape and displaying functionalgroups packed closely together at the surface. Recently, therehas been a series of papers describing how these particlesmight be used for drug delivery applications [22-24], as theiraccessible chemistry and unique architecture potentially ena-ble the encapsulation and release of guests held within theircore to be controlled with some precision [25,26]. Dendrimerswith hydrophilic groups at the surface and hydrophobic coresare capable of solubilising molecules as hydrophobic as pyreneand, unlike conventional micelles, maintain their structureindependent of concentration and in a range of solvents. Sus-tained release of indomethacin has been demonstrated fromdendrimers composed of a hydrophobic core with PEG-chains at the surface and drug loadings of up to 11 wt% wereachieved [27]. More recent work has extended the applicationsof dendrimers to target DNA delivery [28] and to address thebiocompatibility and biodistribution of commercially availa-ble poly(amidoamine) dendrimers in vivo [29].

4.1.3 Phase-transition polymers for drug deliveryIt has long been accepted that as natural systems exert dynamiccontrol over chemical messaging, so it is necessary in manytherapies that drug release can also be regulated, ideally in

Figure 1. Chemical structures of polymer therapeutics PK1 (1) and PK2 (2).

95 5

Degradable peptide linker

Drug(adriamycin)

Polymer carrier n m p

Cell targeting group(galactosamine)

1 PK1 2 PK2

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response to a biochemical or environmental signal [30]. Recentefforts have focused particularly on responsive or active drugtargeting because even if a drug can normally be liberated at aconstant rate (zero-order release), changes in physiologicalcycles or metabolism may alter the threshold concentration in,for example, the bloodstream such that the drug is no longer atits optimum level. There is thus a need for mechanisms to bebuilt into delivery vehicles such that they can respond, on aphysiological time-scale, to an external stimulus in order torelease the pharmacologically active agent at exactly therequired time. As a consequence, a large variety of responsive(smart) polymers have been developed and are now beinginvestigated for pharmaceutical applications on account oftheir ability to display a phase transition dependent on achange in environment [31,32]. Typically, these polymers are sol-uble in aqueous media at a given pH, ionic strength or temper-ature but become insoluble under certain critical conditions. Anumber of examples are shown in Figure 2.

An important criterion for these novel drug delivery vehi-cles is the non-linearity of their response and thus considera-ble effort has been expended in developing polymers thatdisplay very sharp transitions for a small change in environ-ment. Perhaps the most-studied polymer in this regard ispoly(N-isopropylacrylamide) (PNIPAm or PNIPAAm),which is soluble in aqueous media below 32C but which rap-idly precipitates from solution above this temperature [33,34].This phase behaviour results from the entropic gain of watermolecules being released into the bulk aqueous phase over-coming the enthalpic contribution to the system of waterhydrogen-bonded to the polymer chain. The temperature atwhich this occurs (the lower critical solution temperature orLCST) is largely dependent on the hydrogen-bonding capa-bilities of the constituent monomer units and thus the LCSTof a given polymer can in principle be tuned as desired. As aconsequence, a very large number of temperature-sensitivematerials based on co-polymers of PNIPAm with monomersvarying in hydrophilicity or hydrophobicity have now beenprepared [35,36], as well as derivatives of poly(ethylene oxide)-co-poly(propylene oxide) [37] and substituted celluloses [38],which also display temperature-sensitive phase behaviour.

When PNIPAm is incorporated into a cross-linked poly-mer, the resultant gel can exhibit thermally-reversible shrink-age or collapse above the LCST of the homopolymer. Thischange in the structure of the matrix (known as the lower gelcollapse point) is accompanied by loss of water and any co-

solutes and thus is an attractive mechanism for drug release orreaction control [39-41]. For example, swollen hydrogels basedon PNIPAm, kept in drug solutions at low temperatures, dis-play rapid initial drug release when transferred to a medium attemperatures well above the gel collapse point. This is attrib-uted to a fast contraction of the matrix, expelling the drugalong with water and is accompanied by a slower release pro-file as the drug diffuses from the more compact shrunken gel,as shown schematically for the release of indomethacin (3) inFigure 3. Pulsed release of indomethacin has been achievedfrom PNIPAm-co-butylmethacrylate gels, although a com-plete switch-off in terms of drug release was also observedfrom this matrix at 30C as the polymer collapsed to form animpermeable skin surface [42].

There are clearly many variables which affect the rate ofdrug release from such collapsible hydrogels, including poly-mer solvation and swelling, cross-link density and non-spe-cific or specific interactions of the drug with the matrix. Inaddition, the diffusion or release of the active therapeutic var-ies markedly with polymer elasticity, matrix pore size and sol-vent ingress. The detailed kinetics of drug release fromhydrogels are exceedingly complex and have been the subjectof numerous studies [43-46], however, for certain stimuli-responsive gels, more simple correlations between matrixbehaviour and drug release can be observed. In some cases,the swelling of a collapsed thermosensitive gel containing apre-encapsulated drug can be used as a release mechanismrather than gel collapse and solute expulsion. Alternatively, byattaching PNIPAm to a pre-formed polymer capsule contain-ing a therapeutic, the release of the drug can be switched offabove the LCST and restored when the system is returned to alower temperature. For example, the grafting of PNIPAm to apermeable nylon capsule membrane resulted in a gradualreduction in release of encapsulated napthalene disulfonate,with a sharp decrease in permeability above the LCST [47].

Since these early reports of thermally-responsive hydrogels,various hybrid polymer systems have been developed in orderto enhance the rapidity of gel collapse and thus to obtainmore accurate control of drug release. The grafting of PNI-PAm oligomers to an existing hydrogel has enabled theresponse time of the matrix to be reduced to around 20 mincompared to the extended (> 1 month) time required to col-lapse an ungrafted hydrogel [48]. This is thought to occur bythe rapid desolvation of the shorter-chain oligomers, whichthen provide hydrophobic nuclei onto which the remaining

Figure 2. Examples of hydrophilic phase-transition polymers.

H+

Poly(N-isopropylacrylamide)temperature responsive

Poly(vinyl alcohol)-co-poly(acrylic acid)pH and electric field responsive

Poly(acrylic acid)pH responsive

O NH

n

O OH

m

O OHOH

n

m

O OOH

n

m

HO-

Alexander


network aggregates. Although the re-swelling of the matrix isvery much slower than the LCST-induced collapse, this meth-odology is promising for applications where an extremely fastrelease of drug is required after an initial stimulus.

Another innovative approach to controlled drug releaseusing thermally responsive hydrogels has been to incorporatethe polymer into a valve-based device [49]. A disc ofpoly(NIPAm-co-acrylamide) is inserted underneath a cap andan impermeable Teflon cylinder used as a reservoir for thedrug solution. Above the critical temperature, the gel is in acollapsed state, allowing drug diffusion through the cap,whereas below this point, the polymer expands and switchesoff the valve. The simplicity of this device and the ability tochange the temperature at which the valve operates by usingpolymers of various LCST potentially offers very fine controlover drug release.

The possibility of using thermal segregation of polymerchains rather than aggregation or collapse has also beenexplored as a method for controlled drug delivery. Judiciouschoice of monomers, backbone and side chain components interms of their acid base or H-bonding behaviour potentiallyallows the assembly-disassembly temperature of inter- andintra-polymer complexes to be programmed in. In this way, adrug encapsulated in the polymer matrix can be released at apre-determined point dependent on the number and natureof the interchain bonds. Pulsed release of ketoprofen from aninterpenetrating network (IPN) of poly(acrylic acid) (PAAc)and poly(acrylamide) has been effected by heating the IPN toa point above which co-operative H-bonding interactionsholding the chains together begin to break down. The co-pol-

ymer gel becomes more swollen and the drug is released. Thisprocess can be switched off by reducing the temperature tobelow a critical value. The unzipping of the co-polymer net-work is of course analogous to biological polymer-polymerinteractions, such as protein folding and DNA double strandcleavage, and it is likely that bio-inspired polymer engineer-ing of this type will be more greatly used in the future for con-trolled delivery.

4.1.4 Chemically-responsive polymersThe facile synthesis of poly(alkylacrylamide) and PAAc co-polymers readily allows the introduction of components ableto respond to changes in chemical environment, such as ionicstrength and pH. Indeed, PAAc and poly(methacrylic acid)(PMAc) based hydrogels have been amongst the first to bestudied as drug delivery vehicles [50] on account of their abilityto swell reversibly with changes in pH. In addition, the lowcost of acidic acrylic polymers and their adhesion to biologicalsurfaces when partially protonated (see Section 4.2.1) havealso contributed to making this class of polymers of long-standing interest in biomedical applications [51,52].

The swelling of these polymers at neutral or higher pHrenders them attractive for drug-delivery via non-parenteralroutes, such as via the pulmonary or nasal membranes. Thesteroid budesonide (4), related to 16--prednisolone, is a use-ful drug for the treatment of seasonal or allergic rhinitis but ispoorly soluble in water and thus has low bioavailability ifadministered by conventional oral routes. Encapsulation ofbudesonide in cross-linked PMAc and PEG co-polymermicroparticles (5), containing lactose as a compatibiliser, ena-

Figure 3. Release of indomethacin (3) from thermoresponsive polymer gels.LCST: Lower critical solution temperature; T: Temperature.

Desolvatedpolymer gel

Swollen gel

T < LCST

Solution of (3)

T > LCSTImplant

T > LCSTRemove solvent

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bled rapid initial release of the drug, increased bioavailabilityand, compared to budesonide administered intravenously, amore sustained diffusion profile after the primary burst [53].

The fast initial release was attributed to rapid gel swellingof the co-polymer matrix after entering the nasal cavity andthe near constant concentration of budesonide detected inplasma for at least 8 h attested to the subsequent gradual (Fic-kian) diffusion of steroid from the gel through the nasalmucosa. The desirability of non-parenteral administration islikely to ensure that many more pH sensitive hydrogels aredeveloped for specific applications via oral or nasal routes [54].

Perhaps not surprisingly, there have been a number ofreports where both temperature and pH sensitive moietieshave been incorporated into polymeric drug delivery vehicles.A co-polymer prepared from N-isopropylacrylamide andmethacrylic acid has been used to control heparin and strep-tokinase release, as a potential mechanism for controlleddelivery of antithrombotic agents to blood clots [55]. AlthoughpH and temperature response of this system in terms of rapidtherapeutic release was limited, sharp transitions of matrixswelling dependent on changes in solution pH and tempera-ture were observed and drug release could be correlated withthe overall swelling of the matrix. Related hydrogels of PNI-PAm and PAAc have also been used to control the release ofcalcitonin in response to acidity and temperature changes [56],with the gel in its collapsed state providing protection for theprotein therapeutic under acidic conditions whilst enablingrelease at higher pH after matrix swelling.

However, there can be difficulties in predicting the pHresponse of polyelectrolytes in complex ionic media (forexample biological fluids) and thus there are still a number offundamental design criteria which need to be established forthe use of acid- or base-sensitive polymers in vivo. A recentexample is the demonstration that the acid-base behaviour ofco-polymers of N-isopropylacrylamide with N-methacryloyl-L-leucine is different at the LCST than when the polymeradopts a random coil formation [57]. Consequently, dependent

on the degree of ionisation of the leucine residues (which willbe influenced by the local environment), the polymer may notundergo a phase transition at the desired temperature andthus release a drug at the correct site.

In parallel with the phase-transition approach describedabove, a number of specialised polymer drug delivery systemshave been developed, which can respond only to a highly spe-cific molecular signal. A particular goal has been a glucose-sensitive insulin release system for diabetes treatment andphase-transition release linked to glucose concentration hasobvious therapeutic advantages. One approach employed glu-cose oxidase immobilised in a hydrogel composed of 2-hydroxyethylethacrylate (HEMA), N,N-dimethylaminoethyl-methacrylate (DMAEMA) cross-linked with a hydrophilicmonomer, tetraethyleneglycoldimethacrylate (TEGDMA).When glucose was able to permeate the gel and reach glucoseoxidase, it was converted to gluconic acid and the concurrentlocal reduction in pH caused swelling of the gel as theDMAEMA residues were protonated. As a result, the permea-bility of the gel to insulin was increased and by varyingDMAEMA content, cross-linking ratio and pH, a co-polymerwith phase transitions and responses at a sensitivity range of 0- 100 mg/l glucose was achieved [58]. Variation of glucose oxi-dase levels from 0.1 - 2.0 wt% (relative to the polymer scaf-fold) did not however influence the swelling behaviour of thehydrogel with different glucose concentrations, although theaverage permeability of insulin did increase, by up to 5.5-fold,after addition of glucose [59]. Moreover, a significant disadvan-tage of this system was insulin leakage, which would clearlyhave severe medical implications. As a consequence of theseconcerns, devices have now been developed where both insu-lin and glucose oxidase were uniformly distributed through-out a solid polymer matrix. Incorporation of DMAEMA athigh loading (18.5 wt%) and a low cross-linking ratio (0.3vol%) enhanced the swelling and release kinetics and enabledthe hydrogel to display sensitivity to glucose at physiologicalconcentrations. Nevertheless, mass transfer limitations and

Figure 4. pH-mediated release of budesonide (4) from poly(methacrylic acid-co-PEGmethacrylate) (5) hydrogels.

H2O

pH 7-8nasal cavity

Budesonide release

H2O/EtOH

pH 7.3

4

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O

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enzyme deactivation limited the performance of the matrixand the gel displayed rather slow response times in terms ofinsulin release to changes in glucose concentration [60]. Asoxygen is only poorly soluble in aqueous solutions, the con-version of glucose to gluconic acid becomes rate limited byoxygen availability in the polymer microenvironment and thisconsequently limits the rate of matrix swelling and insulinrelease. Co-incorporation of catalase into the polymer matrixenables the generation of oxygen which is then utilised by glu-cose oxidase to convert glucose to gluconic acid. The key reac-tions in these pathways are shown below:

The second reaction has the additional benefit of removinghydrogen peroxide, which otherwise results in product inhibi-tion of the glucose oxidase enzyme. The results from thisstudy demonstrated that enhanced insulin release could beobtained by incorporation of up to 9.4 units of catalase toglucose oxidase and that in simulated in vivo conditions (i.e.,with a steady-state concentration of oxygen in the externalsolution), the swelling of the matrix, as measured by wateruptake, increased by factors of two to five within 200 min.Implantation of these membranes in rats showed that at leastsome of the immobilised insulin retained its activity and a sig-nificant corresponding decrease in blood sugar levels wasobserved [61]. A similar system was studied by Podual et al.[62], who prepared cationic hydrogels containing immobilisedglucose oxidase and catalase in a poly(DMAEMA-co-PEG-methacrylate) matrix cross-linked with TEGMA. This systemhad the additional advantage of the hydrophilic PEG constit-uent, which is known to impart biocompatibility [63] and theresultant gels displayed pH dependent swelling behaviour,with a transition between swollen and collapsed states at pH7.0. In this case, the changes in mesh size for the matrixbetween the two states were rather small (68 - 72 , over atime period of 15 min) and the hydrogel displayed fasterswelling than collapse. However, the reversibility of the phasetransition was demonstrated, indicating the promise of suchmaterials as glucose-sensitive agents.

The use of acidic co-polymer hydrogels for control of insu-lin release has also been investigated, where the production ofgluconic acid by in situ oxidation of glucose results in a con-traction, rather than a swelling, of the polymer matrix [64]. Inthis case, a chemical valve approach was adopted by immobi-lising an acidic co-polymer conjugated with glucose oxidase atpre-formed surfaces, forming a physical barrier to insulintransport. Introduction of glucose resulted in a shrinking ofthe immobilised co-polymer, enabling insulin to permeate

through the polymer gate. This method has the advantagethat insulin can be contained in a separate reservoir, obviatingthe problems of matrix leakage, although oxygen sensitivityand product inhibition by hydrogen peroxide are still poten-tial problems.

An ingenious method for producing chemically sensitiveco-polymers for insulin release has recently been published,employing a glycopolymer-concanavalin A conjugate [65,66].In this system, insulin is physically entrapped in a matrixassembled from a glucose-containing polymer and concanava-lin A, which contains four binding sites for glucose (and man-nose). Introduction of free glucose into solutions containingthe glycopolymer-lectin-aggregate results in competition forthe concanavalin A binding sites between glucose and theglycopolymer, which at high concentrations of free glucosecauses disassembly of the aggregate and release of insulin.When the concentration of glucose decreases, the glycopoly-mer can once again bind to the lectin and re-entrap insulin.Whilst there are still many issues to be resolved in terms ofkinetics of insulin release, reversibility of aggregate formationand the inherent long-term stability of such a system, the useof selective lectin-mediated chemical triggers with their associ-ated fast on-off binding rates is potentially very promising.

The development of more generic chemically responsivepolymers, for example systems that can change state depend-ent on the presence of a particular enantiomer of a chiralcompound, is also of very considerable interest. For example,the presence or excess of a particular chiral drug might beused to switch on or off a polymer-drug delivery system, thusensuring either an optimal initial dose at the target, or aresponse-release profile to match the therapeutic requirementat the site.

The response of acrylamide co-polymers containing opti-cally active monomers to solutions containing an excess of oneenantiomer of an amino acid (a chiral environment) has beeninvestigated [67]. A polymer containing 50% N-isopropylacry-lamide and 50% N-(S)-sec-butylacrylamide showed a markeddifference in LCST dependent on whether L- or D-tryptophanwas present. In aqueous solution, the co-polymer displayed acloud-point at 23C, which was lower than the LCST of PNI-PAm homopolymer (32C) as expected owing to incorpora-tion of the hydrophobic sec-butyl co-monomer. However,addition of 125 M L-tryptophan raised the LCST of poly(N-isopropylacrylamide co-N-(S)-sec-butylacrylamide) (6) to34.5C, whereas in the presence of D-tryptophan, the polymerbegan to phase separate at 28.7C. Correspondingly, a co-poly-mer containing 52% N-isopropylacrylamide and 48% N-(R)-sec-butylacrylamide (7) exhibited a higher LCST if the solu-tion contained D-tryptophan rather than L-tryptophan.

The difference in LCST of each polymer in the chiral envi-ronment was attributed to hydrophobic - interactionsbetween the aromatic tryptophan side chain and the pendantsec-butyl groups, although a detailed mechanism to accountfor the unusual phase behaviour has yet to be established.

Nevertheless, whilst the interactions even of relatively simple

Glucose + O2 + H2OGlucose oxidase

Gluconic acid + H2O2

2H2O2Catalase

O2 + 2H2O



chiral side chain polymers with solutions containing an excessof individual enantiomers are still poorly understood, the impli-cations for regulated (or feedback-controlled) drug delivery viaa chiral stimulus are clear and this field is attracting growinginterest [68-70]. Whilst a truly biomimetic response is still to bedemonstrated, it is clear that the preparation of a functionalgroup sensitive polymer release system is achievable and anapplication would find many uses in therapy.

4.2 Cell-specific targeting4.2.1 Bioadhesive polymers for drug deliveryThe concept of using polymers that are capable of attachingto biological surfaces for the purpose of drug delivery was firstdescribed in the early 1980s [71,72], with the primary aim ofincreasing the residence time of drugs at the intended site ofaction. These polymers are generally described as mucoadhe-sive, owing to their ability to bind to mucosal surfaces and areable to increase the rate of absorption of drugs at epithelialbarriers. The advantages of mucoadhesive polymer systems forcontrolled drug delivery are now well-established and havebeen the subject of numerous publications and reviews [73-75].However, because these polymers attach to the mucus layer,which is itself a complex polymeric aggregate and constantlyundergoing turnover, the controlled release of a therapeuticagent to the desired site can be compromised. As a result,there has been intensive effort in developing polymers thatcan attach to particular cells (cytoadhesion) rather than themucosal layers in order to enhance the specific targeting of theadministered drug. This requires the polymer to recognisethe desired cells via selective chemical interactions andbecause cell surfaces contain complex glycosylated membranecomponents, which are also subject to dynamic changein vivo, this presents a formidable challenge.

4.2.2 Lectin-mediated deliveryOne approach is to use polymer-lectin conjugates, exploitingthe sugar-binding specificity of the lectin to attach the poly-mer delivery vehicle to the cell and then trigger cross-mem-brane transport processes. A significant potential advantage ofusing lectin- or bacterial invasin-mediated recognition is thatthe signal transmitted to the cell on binding may not onlyresult in adhesion but may also trigger cell internalisationpathways, further enhancing the efficiency of drug delivery.

Wheat germ agglutinin (WGA), lycopersicon esculentumagglutinin (LEA) and urtica dioica agglutinin (UDA) labelledwith fluorescein isothiocyanate (FITC) have been shown tobind strongly to Caco-2 cell monolayers and at 37C, FITC-UDA and FITC-WGA showed reduced fluorescence, indicat-ing internalisation of the lectins into the acidic (FITC-fluores-cence quenching) domains within the cell. The correspondingrhodamine-labelled lectins, which exhibit pH-independentfluorescence, were shown to retain their fluorescence insidethe cells by confocal laser scanning microscopy. Conjugationof WGA to liposomes enabled binding to human alveolar cellsand this binding could be reversibly inhibited by addition ofN-acetylglucosamine oligomers, which are ligands for WGA.The specificity of WGA liposome cytoadhesion was demon-strated by addition of serum albumin and by a synthetic lungsurfactant Alveofact (bovactant, Boehringer Ingelheim),both of which failed to inhibit binding of the lectin-liposomeconjugate to the cell surface. In addition, confocal laser scan-ning microscopy indicated internalisation of WGA-liposomesinto primary human alveolar cells and release of FITC-dex-tran from the liposomes into alveolar epithelial cells [76]. Asimilar approach, employing nanoparticles coated with a bac-terial invasin fusion maltose binding protein (INV-MBP)showed a 2- to 3-fold increase in binding to canine kidney

Figure 5. Variation of phase transition with chiral environment: poly(N-isopropylacrylamide-co-N-sec-butylacrylamide) (6,7)LCST in the presence of L-tryptophan (8) and D-tryptophan (9).

6 LCST 23C

+

8 L-Tryptophan

9 D-Tryptophan

34.7C

LCST

28.7C

7 LCST 23C

8 L-Tryptophan

9 D-Tryptophan > 30C

< 30C

O NHO NH

52 48

O NHO NH

50 50

+

(S)

(R)

(S)

(R)

(R)

(S)

NH

O

OH

NH2

NH

O

OH

NH2

NH

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OH

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OH

NH2

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epithelial (MDCK) cells compared to the same nanoparticlescoated with a control maltose binding protein [77]. This bind-ing, due to interaction of invasin with 51 integrins on thesurface of the MDCK cells, was completely inhibited at 4C,which was attributed to a loss of cell membrane fluidity andcorresponding blocking of the integrin-invasin interaction atlower temperatures.

4.2.3 Synthetic polymer lectin mimicsAlthough polymer-lectin conjugates exhibit a broad spectrumof medicinally valuable properties, the very high cost of somenatural lectins and their potential immunogenicity has ledmany researchers to consider alternatives. A highly promisingapproach, which is soon likely to be adopted for drug deliveryapplications, is the targeting of specific biological surfaceswith multi-valent synthetic polymers. These materials can beconsidered as analogous to lectins in that multiple weak bind-ing processes, typical of sugar-lectin interactions, are utilisedin a co-operative fashion to achieve an overall tight recogni-tion of the biological target by the synthetic polymer. Naturallectin carbohydrate binding domains (CBDs) employ hydro-gen-bond donation from protein amide groups to oxygenlone-pair acceptors on the target sugars and hydroxyl groupH-bond donation from the carbohydrate to carbonyl accep-tors on the lectin [78]. In addition, specifically bound watermolecules and hydrophobic interactions at apolar sites on thesugar molecules contribute to a multitude of binding interac-

tions thus invoking high specificity [79]. Synthetic polymersare now being designed which have similar multiple H-bonddonors, acceptors and hydrophobic domains, the aim being todevelop a drug delivery vehicle that can bind cell-surface sac-charides with high specificity irrespective of whether the sugarresidue has a suitable or easily accessible natural receptor.

The successful demonstration of the principle behind thisapproach involved the preparation of neoglycopolymers andtheir competitive inhibition of erythrocyte agglutination bythe glucose/mannose binding lectin concanavalin A. Com-pared to the monomeric sugars (10a,b and 11a,b), the glycopoly-mers (12,13) showed up to 50,000-fold enhancement ofconcanavalin A inhibition and C-glycoside polymers weremore effective than O-glycoside derivatives. The latter increasein specificity was attributed to the greater hydrophobicity ofthe C-glycosyl residues, which accords well with the hypothesisthat sugar recognition in lectin CBDs is amplified by van derWaals interactions between aromatic side chains of protein res-idues and hydrophobic patches on the bound saccharide.

A further and particularly elegant exposition of the biomi-metic strategy for developing polymer therapies was reportedby Whitesides and co-workers in the search for novel antiviralagents [80-82]. The influenza virus is able to invade cells byexpressing multiple copies of haemagglutinin (HA) on itsouter coat, which binds strongly to N-acetylneuraminic acid(sialic acid) on host cell surfaces. The virus also displays neu-raminidase (NA) at its surface, which acts to release the virion

Figure 6. Inhibition of concanavalin A-mediated erythrocyte agglutination by synthetic neoglycopolymers (12,13).

Sugar derivative Relative inhibitory dose

Glucose derivatives(10a and 10b)

R = CH31.0R = CH2CH=CH20.5

Mannose derivatives(11a and 11b)

R = CH30.25R = CH2CH=CH20.5

0.04O-glucoseneoglycopolymer (12)

0.00001C-mannose neoglycopolymer (13)

O

OH

OHOH

OH

R

O

O

OH

OHOH

OH

O OH

OHOH

OH

O

O

O

O

O

OH

OHOH

OHR

O

O

O

OOH

OHOH

OH

O OH

OHOH

OH

O

O

O

O



from the invaded cell by catalysing the breakdown of sialicacid: this process then liberates the virus promoting furtherinfection. Influenza can be potentiated either by blocking theattachment of virus cells (HA inhibition) or by preventing therelease of the virion (NA inhibition), therefore syntheticagents which bind HA and NA are attractive therapeutic tar-gets. Polymers with both number and sequence diversity offunctional (binding) groups were prepared by combinatorialor multi-parallel substitutions on a pre-formed poly(acrylicanhydride) precursor, thus generating polyvalent inhibitorsbearing multiple pendent sialic acid derivatives. In effect thisgenerated an antiviral library, which was then screened foractivity in assays measuring inhibition of influenza virusmediated haemagglutination of chicken erythrocytes. Themost potent polymers (14,15, Figure 7) displayed exceptionallyhigh activity: the minimum concentrations of these syntheticneoglycopolymers required to inhibit viral-mediated haemag-glutination were as low as 10-11 M, corresponding to a 1000-fold enhancement of activity compared to the most potentnatural inhibitors.

The generality of this method and its relative simplicityoffers the possibility of preparing materials that could be usedto attach a therapeutic agent directly and specifically to poten-tially any desired biological target. For example, further con-jugation of a particular therapeutic would allow directdelivery to the target, whilst analogous linking of a cell- or

virus-binding polymer with a specific inhibitor should enableselective blocking of undesirable cell or viral action.

4.3 Intracellular delivery and gene therapyPerhaps the most challenging goal for new polymer therapeu-tics is not only to deliver a drug to a particular cell but alsoactively to transport it across the cell membrane into the cyto-plasm and ultimately to the nucleus itself. The enormouspromise of genetic-based therapies [83], whereby a defectivegene can be repaired or replaced by a normal DNA sequence,has to date been compromised by the difficulties associatedwith successful cell transfection by an appropriate vector.Whilst natural viral vectors are able to cross cell barriers withrelative ease, the well-documented problems with potentialpathogenicity or immunogenicity renders a synthetic DNAvector highly desirable.

Pioneering studies in this field involved the use of cationicpolymers, lipids and liposomal carriers [84-88]. Polymers suchas poly(L-lysine) (PLL) and poly(ethyleneimine) (PEI) havebeen extensively evaluated as DNA delivery systems onaccount of their accessibility and net positive charge. Com-plex formation with DNA occurs readily and the resultingmacromolecular aggregates are believed to cause transfectionvia either cationic or hydrophobic binding to cell membranesand subsequent cytoplasm entry. The degree of transfectioncan be partially correlated with cytotoxicity, as non-toxic low

Figure 7. Synthesis of polyvalent haemagglutination inhibitors (14,15).

, AIBN

R-NH2 / H2O

R1-NH2, R2-NH2 / H2Oultrasound, H2OpH 7-12

R = NH2 COOH NH2COOH

R3 = (CH2)3S(CH2)2NH2, O(CH2)2O(CH2)2NH2,

O

O

O O

O

O

O

O

O

R2HN OO

OH O OH O

R1HN

RHN O

OH O

14

15

, ,

OO

NH

NH2

O

NH

SO

O

N

,

NH

NH2O

S

O

NH

OH O R3

OH

O

OHOH COOH

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Figure 8. Structures of gemini-surfactants (16-20) for DNA-transfection.

16 17

RHNNH

SS N

H

NHR

OH

NH

NH2

OH

NH

OHNH2

NH2

O

O

O

O

O

OO

O

NH2 OH18 19

20

R = CH2(CH2)10CH3

NHR

O

RHN

NH2

NH

NH

SS

NH

NH

NH2

NH

NH2

NH2

NH

OH

OH

NH2

NH2

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O

O

O

O O

O

NHR

O

RHN

NH2

NH

NH

SS

NH

NH

NH2

NH

NH2

NH

OH

OH

NH

NH2

O

O

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O O

O

NH

NH2

NH2

O

NH2

NH2

O

NH2

NH2

NH O

NH2

NH2

NHR

O

RHNNH

SS N

H

OH

OHO

O

O

NHO

NH O

NH2

NH2

NH2

NH2

RHNNH

SS N

H

NHR

OH

OHO

O

O

ONHO



molecular weight PLL species display little or no transfection,whereas high molecular weight PLL causes severe membranedisruption but high transfection. It is likely therefore that acompromise will be required for the use of such polymers ingene therapy to enable DNA transport to the nucleus to occurwithout causing excessive cell damage [89].

Lipid vectors have a useful combination of properties suchas low toxicity, ready availability and low immunogenicity.Polyelectrolyte interactions between negatively charged phos-phate groups on the DNA backbone with cationic lipids leadto complex formation, with hydrophobic lipid groups ori-ented away from the charged centre. The resulting species arehydrophobic and poorly soluble in water but are able to inter-act with cell membranes and are effectively taken up intomembrane vesicles, possibly via disruption of the membranesby the lipophilic portions of the complex [90,91]. The mostimportant variables governing DNA delivery via cationic lip-ids and liposomes are postulated to be the net charge of thecarrier and the presence of membrane lipid bilayers abovetheir melting transition, indicating that relatively simple fac-tors can markedly affect rather complex biological processes.However, lipid-DNA complexes suffer from the disadvantagethat the polynucleotide is susceptible to degradation if notstrongly bound: although this can be circumvented to someextent by the encapsulation of DNA within a liposomal mem-brane, the inherent technical difficulties in liposome prepara-tion and the potential size limits of the DNA which can beincorporated are still issues to be resolved.

Lower molecular weight or oligomeric delivery vehiclescapable of membrane disruption are also of interest, as thesecan potentially be prepared with much greater precision thanpolymeric carriers or liposomes. In particular, peptide basedsurfactants show considerable promise and the gemini sur-factants (16-20, Figure 8) (detailed in patent applicationWO9929712, SmithKline Beecham plc) are currently under-going trials for DNA delivery [92]. Use of a co-lipid and addi-tion of a basic polypeptide markedly increased transfectionefficiency of these surfactants [93]. In addition, as these speciescan be prepared by adaptation of conventional peptide chem-istries, a wide range of structures are accessible to synthesisand characterisation by high-throughput methods.

In common with liposome and peptide surfactant deliveryvehicles a major focus of polymer research is the developmentof synthetic polymer-DNA conjugates that are designed notonly to cross the cell membrane but also to destabilise intrac-ellular barriers enabling release of the gene into the nucleus.For example, poly(ethylacrylic acid) (PEAAc, 21), has beenshown to display pH dependent lipid vesicle disruption [94]and in red blood cell haemolysis assays PEAAc of molecularweight 26 kDa proved to be more effective than mellitin, apeptide of noted membrane disruptive activity [95]. A relatedpolymer, poly(propylacrylic acid) (PPAAc, 22), is even moreactive than PEAAc and results in a 15-fold higher efficiency ofred blood cell haemolysis, on account of the more hydropho-bic isoleucine analogue backbone [96].

Combinations of PPAAc with PLL have been evaluated fortransfection of green fluorescent protein into NIH3T3 cellsusing the pEGFP plasmid as DNA source. Low transfectionefficiencies were reported for PPAAc alone but prior particleformation of PPAAc with PLL and co-incubation with PLL-plasmid complexes resulted in 20-fold enhancement of celltransfection. This indicates that PPAAc is effectively taken upby the cells and can subsequently act as a membrane disrupt-ing species following a reduction in the endosomal pH.

Conjugation of biotinylated PPAAc with streptavidinafforded a polymer-protein complex (Figure 9b) that retained itshaemolytic activity, suggesting that the biological action of thepolymer is not compromised by the presence of the protein.

In addition to membrane disruption, cell surface targetingmay also be used to enhance internalisation of DNA and theintroduction of specific molecular recognition moieties whichmediate endocytosis, such as fibroblast growth factors (FGF)and vascular endothelial growth factor (VEGF) receptors, areattracting much interest. Incorporation of the tripeptide Arg-Gly-Asp (RGD), which binds to V and 5 integrins,increases transfection of cells in vitro via a promotion of inter-nalisation following integrin binding, in a manner believed tobe similar to that which occurs with adenoviruses [97]. Strepta-vidin mutants have been prepared which contain surface-dis-played RGD and these have been shown to bind strongly torat aortic endothelial cells, whereas wild-type streptavidin andcontrol tripeptides showed no binding above background lev-els. It is to be expected that further conjugation of the strepta-vidin-RGD constructs with biotinylated PPAAc will displayenhanced cell surface binding in addition to increased mem-brane disruption and it is highly likely that this combinationof targeting and delivery systems in one vehicle will prove veryeffective in gene therapy applications.

The potential pH-responsive nature of the above polymersis, by analogy with other drug delivery systems, of interest as amethod for switchable DNA delivery. It is not surprisingtherefore, that the structures which confer temperature and orpH response in conventional aqueous polymers, such as PNI-PAm, PPAAc and PDMAEMA, are now being adapted forapplications in gene therapy. In addition, responsive polymersoffer one very significant potential advantage over conven-tional polymeric DNA vectors: the change in the polymerstructure can be used to complex the genetic material in areversible manner. In this way, the polymer affords protectionagainst nucleases during transport to and across cell barriers inone state and can then be switched to another state to releasethe DNA into the cell for transcription by RNA polymerases.It is this ability to protect, transport and release DNA, all ofwhich are essential if successful transfection is to occur, whichmakes thermo-responsive polymers especially attractive asnew vectors.

The most actively investigated responsive polymer systemsfor potential gene therapy applications are those preparedfrom N-isopropylacrylamide and N,N-dimethylaminoethyl-methacrylate (PNIPAm-co-PDMAEMA). The cationic

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DMAEMA moieties provide a means for complexing DNAwhilst the PNIPAm chains confer temperature-dependent sol-ubility behaviour. DNA binding has been observed for co-polymers containing as little as 15 mol% DMAEMA, suggest-ing that highly charged (and thus potentially more cytotoxic)polymers are not necessarily required for effective transport ofpolynucleotides [98]. In this study, complexation of pCMV-LacZ plasmids with PNIPAm-co-PDMAEMA polymers ofvarying molecular weights and monomer contents was fol-lowed by incubation of the aggregates with OVCAR-3 cells.Monitoring of transfection efficiency by an X-Gal (-galactos-idase activity) assay indicated that transfection took place butwas strongly dependent on a number of experimental varia-bles. In general complexation of the co-polymers with DNAat 25C was accompanied by precipitation as the overallLCST of the polymer conjugate decreased and particles of~ 200 nm diameter were formed at higher co-polymer:plas-mid ratios indicating condensation of the plasmid within thecomplex. For low molecular weight co-polymers bound to

DNA, aggregation to larger particles took place at 37C (i.e.,above the LCST of PNIPAm) whereas the higher molecularweight co-polymers or those with greater NIPAm contentwere relatively stable at the higher temperature and thesemore stable 200 nm complexes proved most effective at genetransfection. In addition, the cytotoxicity of the co-polymersdecreased with increasing NIPAm content, which was corre-lated with decreasing zeta potential of the co-polymer-DNAcomplexes and this was accompanied by a correspondingdecrease in transfection efficiency. These results are in accordwith previous studies of cationic DNA-delivery vehicles inthat the properties required for efficient transfection must bebalanced with the associated increasing cytotoxicity (due pre-sumably to increased membrane disruption) that highlycharged species confer.

One method by which transfection efficiency might beincreased other than by increasing the overall positive chargeof DNA delivery vehicles is to incorporate hydrophobic resi-dues into the co-polymer backbone. Recent work has demon-

Figure 9. a) Structures of membrane-disrupting pH switchable polymers poly(ethylacrylic acid) (21), poly(propylacrylic acid)(22) and b) schematic of polymer-biotin-streptavidin conjugate (23).

b

a

NHS-LC-BiotinAmine-terminatedPPAAc Biotinylated PPAAc Streptavidin

23 PPAAc-Biotin-streptavidin conjugate

21 Poly(ethylacrylic acid)PEAAc

22 Poly(propylacrylic acid)PPAAc

O OH

n

O OH

n

O OH

NH2n

NHNH

S

O

O

O

N

O

O O OH

NH

O

NH

NH

S

O

(CH2)4

n



strated that PNIPAm-co-PDMAEMA polymers containingup to 11 mol% of a third monomer, butylmethacrylate(BMA), exhibit enhanced transfection of COS-1 cells (SV-40transformed African green monkey) with the pCMV-LacZplasmid [99,100]. This was attributed to the effect of the extrahydrophobic component on the stability of the co-polymerDNA complex and possible enhancement of cell-surfacebinding prior to subsequent endocytosis. Furthermore, it wasreported that the same co-polymer system displayed tempera-ture-dependent DNA association-dissociation and that byreducing the temperature of incubation of the co-polymercomplex with COS-1 cells to below the LCST of the co-poly-mer, gene transfection increased. This latter result is of con-siderable significance, as it suggests that appropriate choice ofco-monomers and control of polymer composition and struc-ture can be used ultimately to regulate the release of DNA forgene transfection. Whilst this research is still in its infancy, itis nevertheless of considerable potential benefit as a methodfor controlled gene delivery.

Perhaps the ultimate target of non-viral gene therapyresearch involves the combination of cell-targeting, nuclearlocalisation and DNA release systems all in one polymer con-jugate. The general features of such a polymer-DNA vectorare shown diagramatically in Figure 10.

The synthesis of such a vector represents a considerablechallenge, as the polymer must evade all natural barriers (non-specific protein adsorption, enzymatic degradation andimmune response) prior to arrival at the cell, then attach spe-cifically to the target surface, enter the nucleus and finallyrelease the plasmid DNA. It is also not clear whether such alarge and complex conjugate could effectively cross cell mem-branes, or if the release of the plasmid from the polymerwould lead to successful transfection in the presence of all theother components. However, the development of the firstsuch conjugates is already under way in a number of laborato-ries and it is likely that this will be a very active area ofresearch in the immediate future.

5. Competitive environment

Controlled drug delivery is amongst the most active areas ofresearch worldwide, with web-based sources (e.g., Drug Dis-covery Online, Pharmalicensing), indicating that the numberof industrial and academic laboratories active in this field cur-rently (June 2001) stands at over 900 companies and 250 aca-demic groups. Polymeric drug delivery vehicles are also

generating much interest, with new polymer-based releasetechnologies for pain management (Amarin Corporation)hydrophobic drug delivery (ImaRx Therapeutics) and humangrowth hormone receptor antagonist (Shearwater Polymersand Sensus Drug Development) all being announced in thelast 12 months. Within the academic environment, researchinto smart and bioadhesive polymers is equally competitive(for example nearly 50 papers on these subjects alone havebeen published in primary journals over the period December2000 - June 2001) and the underlying interdisciplinarity ofthe area is attracting growing numbers of researchers from tra-ditionally very different subject backgrounds. However,despite the intensity of work in polymeric drug delivery sys-tems, there is still much scope for further research in this field,as a great variety of medical conditions await better control ofdrug release and targeting in vivo. A key example is the use ofplatinum compounds in anticancer treatments, which is still atherapy of choice in many cases despite the well-documentedproblems with small-molecule cytotoxic agents. Polymer-based anticancer therapeutics which do not present these sideeffects are of very obvious medicinal value and as the currentvalue of platinum compounds in cancer treatments is aroundUS$800 million per year, the market potential of such a poly-mer anticancer vehicle is high.

6. Expert opinion

Although the latest generation of polymer drug delivery vehi-cles, particularly polymer therapeutics such as PK1 and PK2,are highly sophisticated medical agents in themselves, formost commercial applications involving polymeric drug deliv-ery, formulations based on poly(ethylene oxide) and acrylicand methacrylic acids are most likely to be adopted at the out-set as these have already found both industrial and clinicaluse. Nevertheless, even for polymers derived from the aboveclasses, there are still a great many variations in structures,properties and functions that have still to be explored. Theflexibility inherent to synthetic chemistry enables a vast arrayof structures with a corresponding range in properties to beproduced and in the laboratory these are limited only by thecreativity and ingenuity of the chemist.

Preparative organic chemistry has now reached a pointwhere molecules with highly complex architectures can beproduced in significant quantities and their structures probedin detail by a huge variety of analytical techniques. Until rela-tively recently, the same was not true of synthetic polymer

Figure 10. Schematic of optimal synthetic vector for DNA delivery.

++

++ Cell surface

recognitiongroup

Sacrificiallinker forDNA releaseNuclear

localising sequence

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chemistry, in that fine control over macromolecular architec-ture was only achievable for a relatively small number of sys-tems and characterisation of polymer structures wasinherently limited to the study of the resulting mixtures.However, recent advances in controlled polymer synthesis,especially those involving living free-radical procedures [101-105], are likely to result in much greater precision in polymerpreparation and a convergence between those structures previ-ously classed as polymers, oligomers and macromolecules.The combination of these free-radical-based strategies withother controlled polymerisation methods (anionic [106], cati-onic [107] and metathesis [108,109]) is already allowing syntheticpolymer chemists to design and prepare macromolecules oftailored molecular weights and with a large variety of func-tional groups previously inaccessible by other routes. In paral-lel the commercial availability of synthetic dendrimers isenabling scientists from outside the discipline of polymerchemistry to use synthetic macromolecules, with biomimeticproperties in terms of size, dispersity and chemical reactivity,in biologically relevant applications [110]. Therefore, as thepolymer chemists toolkit expands, so too will the potentialapplications for the resulting new materials and chief amongstthese will be the next generation of drug delivery vehicles.

One aspect that should be noted, however, is that manyresearch polymers face considerable practical and theoreticalbarriers before clinical use can be envisaged. Phase transitionpolymers in particular are still to be developed fully for drugdelivery applications, as the factors determining the thermody-namics and kinetics of the coil-globule transition (LCST forlinear polymers) and hydrogel collapse (lower gel collapse tem-perature in cross-linked systems) are insufficiently understood[111]. For example, it is not currently possible to predict exactlywhether a given polymer gel will collapse at a pre-determinedpoint (e.g., pH or temperature change) and thus the develop-ment of responsive polymers might still be considered to be atan empirical stage. In addition, the slow response times andinherent asymmetry of gel expansion and collapse are limitingfactors for the use of these polymers in therapy. However, asnoted above, the increasing control that synthetic chemists areable to exercise over polymer structure means that materialswith much sharper phase transition points and response ratesare likely to become available. This ability to control structurewill also impact significantly on the preparation of cell surfaceand cell nuclei targeted polymers, which offer many importantadvantages over conventional drug delivery vehicles, althoughthe clinical use of these materials has yet to be demonstrated. Itis also important to consider that perhaps the biggest hurdles toovercome are the likely high cost of these increasingly complexbiomimetic synthetic polymers and their eventual fate in vivo.Whilst these questions cannot be answered at such an earlystage in the development of the new generation of biomedicalpolymers, there will necessarily be a compromise between the

advantageous clinical effects of these materials and the econom-ics of their production.

Nevertheless, the scope for synthetic polymers in drugdelivery applications is exceptionally wide, with a vast array ofclinical conditions that require selective targeting of therapeu-tic agents to particular biological targets. As our understand-ing of disease at the molecular level increases, so the ability toprepare drug delivery vehicles which can overcome biologicalbarriers to reach specific sites, then release the therapeuticagent at the right time and in the optimum dose, will becomemore apparent. Conjugation of biological (or biomimetic) celland/or nuclear targeting moieties with biocompatible poly-mers is now within the scope of synthetic chemistry andresponsive release mechanisms are becoming increasingly wellunderstood, enabling more specific and active drug targetingto be accomplished and better, more effective therapies to beadopted. Whilst there are still many obstacles regarding thepreparation of these sophisticated polymers, particularly relat-ing to synthetic difficulty and consequently cost, the genera-tion of artificial materials that have increasingly enzyme-likeproperties in terms of binding specificity and mode of actionis likely to revolutionise drug delivery. In this regard, it isworth noting that a relatively new class of synthetic materials,imprinted polymers [112-116], wherein a cross-linked polymeris assembled around a molecular template or ligand, can dis-play molecular recognition capabilities approaching those ofbiological macromolecules. These materials, which are rela-tively simple to prepare, stable and robust to conditions thatpreclude the use of enzymes, may find use in chemically-trig-gered drug release, where the recognition sites of theimprinted polymer, rather than binding the template used togenerate the imprints, are used to liberate a therapeutic onlyin response to a highly specific molecular signal. Whilst thereare still a number of issues unresolved in terms of the prepara-tion and use of imprinted polymers, such as diversity of bind-ing sites and poor control over matrix structure andmacromolecular architecture, the potential for feedback-con-trol of drug release by such systems is highly promising.

Overall it is apparent that synthetic polymers will soonmake the transition from passive drug release devices intoactive therapeutics and their role as the next generation ofcontrolled drug delivery vehicles seems assured.

Acknowledgements

The author thanks the Engineering and Physical SciencesResearch Council (EPSRC) for an Advanced Research Fellow-ship; the School of Pharmacy and Biomedical Science and theInstitute of Biomolecular and Biomedical Science (IBBS),University of Portsmouth, for a Senior Research Lectureship;and Dr Dariusz Grecki and Dr Simon Young for helpful dis-cussions regarding this manuscript.



BibliographyPapers of special note have been highlighted as either of interest () or of considerable interest () to readers.

1. Technical literature. MacroMed Incorporated, Utah, USA (September 2000).

2. KUMAR MNVR, KUMAR N: Polymeric controlled drug-delivery systems: perspective issues and opportunities. Drug Dev. Ind. Pharm. (2001) 27:1-30.

3. KUMAR MNVR: Nano and microparticles as controlled drug delivery devices. J. Pharm. Pharm. Sci. (2000) 3:234-258.

4. BROUWERS JRBJ: Advanced and controlled drug delivery systems in clinical disease management. Pharm. World Sci. (1996) 18:153-162.

5. HELLER J: Polymers for controlled parenteral delivery of peptides and proteins. Adv. Drug Deliver. Rev. (1993) 10:163-204.

6. PEPPAS NA: Hydrogels and drug delivery. Curr. Opin. Coll. Int. Sci. (1997) 2:531-537.

A useful overview of the types of polymer hydrogel currently being evaluated for controlled release.

7. PEPPAS NA, BURES P, LEOBANDUNG W, ICHIKAWA H: Hydrogels in pharmaceutical formulations. Eur. J. Pharm. Biopharm. (2000) 50:27-46.

A review covering many aspects of drug delivery, including a discussion of the kinetics of release from complex polymer matrices.

8. KAMATH KR, PARK K: Biodegradable hydrogels in drug-delivery. Adv. Drug Deliver. Rev. (1993) 11:59-84.

9. DUNCAN R: Polymer-drug conjugates, PDEPT and PELT: a novel anticancer approach. Clin. Cancer Res. (2000) 6(Suppl.):13.

10. GIANASI E, WASIL M, EVAGOROU EG et al.: HPMA copolymer platinates as novel antitumour agents: in vitro properties, pharmacokinetics and antitumour activity in vivo. Eur. J. Cancer (1999) 35:994-1002.

11. DUNCAN R: Polymer therapeutics for tumour specific delivery. Chem. Ind. (London) (1997):262-264.

12. MATSUMURA Y, MAEDA H: A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the anti-tumour agent SMANCS. Cancer Res. (1986) 46:6387-6392.

The first account of the enhanced

permeation and retention (EPR) effect in anticancer therapy.

13. DUNCAN R: Polymer conjugates for tumour targeting and intracytoplasmic delivery. The EPR effect as a common gateway? Pharm. Sci. Technol. Today (1999) 2:441-449.

A review covering the chemistry and biological action of polymer therapeutics.

14. KOPECEK J, KOPECKOVA P, MINKO T et al.: HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action. Eur. J. Pharm. Biopharm. (2000) 50:61-81.

Primary paper on the effect of antineoplastics bound to biocompatible polymers via enzyme-degradable linkers.

15. OMELYANENKO V, KOPECKOVA P, GENTRY C et al.: Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate. J. Control. Rel. (1998) 53:25-37.

16. SHIAH JG, DVORAK M, KOPECKOVA P et al.: Biodistribution and antitumour efficacy of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude mice. Eur. J. Cancer (2001) 37:131-139.

17. SHIAH JG, SUN YG, PETERSON CM et al.: Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorine(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts. J. Control. Rel. (1999) 61:145-157.

A detailed account of the fate of polymer therapeutics following degradation in vivo.

18. ORLOWSKI S,GARRIGOS M: Multiple recognition of various amphiphilic molecules by the multi-drug resistance P-glycoprotein: molecular mechanisms and pharmacological consequences coming from functional interactions between various drugs. Anti-Cancer Res. (1999) 19:3109-3123.

19. GERMANN UA, CHAMBERS TC: Molecular analysis of the multi-drug transporter, P-glycoprotein. Cytotechnology (1998) 27:31-60.

20. VASEY PA, KAYE SB, MORRISON R et al.: Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents drug-polymer conjugates. Clin. Cancer Res. (1999) 5:83-94.

The first polymer therapeutics to undergo clinical trials.

21. VOGTLE F, GESTERMANN S, HESSE R et al.: Functional dendrimers. Prog. Polym. Sci. (2000) 25:987-1041.

A detailed review of dendrimer science from chemistry through materials science to biology.

22. TWYMAN LJ, BEEZER AE, ESFAND R et al.: The synthesis of water soluble dendrimers, and their application as possible drug delivery systems. Tetrahedron Lett. (1999) 40:1743-1746.

23. WANG D, KOPECKOVA P, MINKO T et al.: Synthesis of starlike N-(2-hydroxypropyl)methacrylamide copolymers: potential drug carriers. Biomacromolecules (2000) 1:313-319.

24. WIWATTANAPATAPEE R, CARRENO-GOMEZ B, MALIK N et al.: Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? Pharmaceut. Res. (2000) 17:991-998.

25. BOAS U, KARLSSON AJ, DE WAAL BFM et al.: Synthesis and properties of new thiourea-functionalized poly(propylene imine) dendrimers and their role as hosts for urea functionalized guests. J. Org. Chem. (2001) 66:2136-2145.

26. PISTOLIS G, MALLIARIS A, TSIOURVAS D et al.: Poly(propyleneimine) dendrimers as pH-sensitive controlled-release systems. Chem. Eur. J. (1999) 5:1440-1444.

27. LI M, KONO K, FRECHET JMJ: Water-soluble dendritic unimolecular micelles: their potential as drug delivery agents. J. Control. Rel. (2000) 65:121-131.

The synthesis and characterisation of PEG-tipped dendrimers, and release profile of entrapped indomethacin.

28. BIELINSKA AU, YEN A, WU HL et al.: Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo. Biomaterials (2000) 21:877-887.

29. MALIK N, WIWATTANAPATAPEE R, KLOPSCH et al.: Dendrimers: relationship between structure and biocompatibility in vitro and preliminary studies on the biodistribution of 125I-labelled polyamidoamine dendrimers in vivo. J. Control. Rel. (2000) 65:133-148.

30. KOST J, LANGER R: Responsive polymeric delivery systems. Adv. Drug Deliver. Rev. (2000) 46:125-148.

Alexander


31. GALAEV IY, MATTIASSON B: Smart polymers and what they could do in biotechnology and medicine. Trends Biotechnol.(1999) 17:335-340.

A useful review covering a number of aspects relating to responsive polymers.

32. HOFFMAN AS, STAYTON PS, BULMUS V et al.: Really smart bioconjugates of smart polymers and receptor proteins. J. Biomed. Mater. Res. (2000) 52:577-586.

33. HESKINS M, GUILLET JE: Solution properties of poly (NIPAAm). J. Macromol. Sci. Chem. (1968) A2:1441.

The first detailed account of the phase transition properties of poly(N-isopropylacrylamide).

34. SCHILD HG: Poly (N-isopropylacrylamide) - experiment, theory and application. Prog. Polym. Sci. (1992) 17:163-249.

Comprehensive review of poly(N-isopropylacrylamide) chemistry and properties.

35. FEIL H, BAE YH, JAN FJ et al.: Effect of comonomer hydrophilicity and ionization on the lower critical solution temperature of N-isopropylacrylamide copolymers. Macromolecules (1993) 26:2496-2500.

36. DAUTZENBERG H, GAO Y, HAHN M: Formation, structure, and temperature behaviour of polyelectrolyte complexes between ionically modified thermosensitive polymers. Langmuir (2000) 16:9070-9081

37. HOFFMAN AS: Environmentally sensitive polymers and hydrogels. MRS Bulletin (1991) 16:42-46.

38. ROSEN O, SJOSTROM J, PICULELL L: Responsive polymer gels based on hydrophobically modified cellulose ethers and their interactions with ionic surfactants. Langmuir (1998) 14:5795-5801.

39. HOFFMAN AS: Applications of thermally reversible polymers and hydrogels in therapeutics and diagnostics. J. Control. Rel. (1987) 6:297-305.

40. CHEN GH, HOFFMAN AS: Graft copolymers that exhibit temperature induced phase transitions over a wide range of pH. Nature (1995) 373:49-52.

Side chain grafts of temperature sensitive polymers to a pH responsive polymer backbone enabling the switching of the copolymers under a variety of conditions.

41. STAYTON PS, SHIMOBOJI T, LONG C, CHILKOTI A et al.: Control of protein-ligand recognition using a stimuli-

responsive polymer. Nature (1995) 378:472-474.

The first example of reaction chemistry control by grafting a thermoresponsive polymer to an engineered streptavidin mutant allowing the on/off switching of the protein binding site.

42. BAE YH, OKANO T, HSU R, KIM SW: Thermosensitive polymers as on-off switches for drug release. Makromol. Chem. Rapid Commun. (1987) 8:481-485.

43. AMENDE T, NIKOS AG: Diffusion controlled delivery of proteins from hydrogels and other hydrophilic systems. In: Protein Delivery: Physical Systems. Sanders LM, Hendren RW (Eds.), Plenum Press, Tokyo, Japan (1997):139-165.

44. CANAL T, PEPPAS NA: Correlation between mesh size and equilibrium degree of swelling of polymeric network. J. Biomed. Mater. Res. (1989) 23:1183-1193.

45. COLOMBO P, PEPPAS NA: Analysis of drug release behaviour from swellable polymer carriers using the dimensionality index. J. Control. Rel. (1997) 45:221-227.

46. NARASIMHAN B, PEPPAS NA: Molecular analysis of drug delivery systems controlled by dissolution of the polymer carrier. J. Pharm. Sci. (1997) 86:297-304.

47. OKAHATA Y, NOGUCHI H, SEKI T: Functional capsule membranes 23. Thermoresponsive permeation from a polymer grafted capsule membrane. Macromolecules (1986) 19:493-494.

48. YOSHIDA R, UCHIDA K, KANEKO Y et al.: Comb-type grafted hydrogels with rapid de-swelling response to temperature-changes. Nature (1995) 374:240-242.

A novel mechanism of hydrogel collapse where pendent linear and flexible PNIPAm chains catalyse the de-swelling of a more constrained cross-linked PNIPAm matrix.

49. DINARVAND R, DEMANUELE A: The use of thermoresponsive hydrogels for on-off release of molecules. J. Control. Rel. (1995) 36:221-227.

The first description of the use of responsive polymers as valve-based drug release devices.

50. BRONDSTED H, KOPECEK J: pH sensitive hydrogels: characteristics and potential in drug delivery. In: Polyelectrolyte Gels. Harland RS, Prudhomme RK (Eds.), ACS Symp. Series (1992) 480:1.

51. KOU JH, AMIDON GL, LEE PI: pH-Dependent swelling and solute diffusion characteristics of poly(hydroxyethylmeth

acrylate-co-methacrylic acid) hydrogels. Pharm. Res. 5:592-597.

52. BRANNONPEPPAS L, PEPPAS NA: Equilibrium swelling behaviour of dilute ionic hydrogels in electrolytic solutions. J. Control. Rel. (1991) 16:319-330.

53. NAKAMURA K, MAITANI Y, LOWMAN AM et al.: Uptake and release of budesonide from mucoadhesive, pH-sensitive polymers and their application to nasal delivery. J. Control. Rel. (1999) 61:329-335.

A detailed account of the release profiles of a therapeutic from an acid-sensitive polymer gel.

54. AIKAWA K, MITSUTAKE N, UDA H et al.: Drug release from pH-response polyvinylacetaldimethylaminoacetate hydrogel, and application to nasal delivery. Int. J.Pharm. (1998) 168:181-188.

55. VAKKALANKA SK, BRAZEL CS, PEPPAS NA: Temperature and pH-sensitive terpolymers for modulated delivery of streptokinase. J. Biomed. Mater. Sci. Polym. Ed. (1996) 8:119-129.

56. SERRES A, BAUDYS M, KIM SW: Temperature and pH-sensitive polymers for human calcitonin delivery. Pharm. Res. (1996) 13:196-201.

57. BIGNOTTI F, PENCO M, SARTORE L et al.: Synthesis, characterisation and solution behaviour of thermo- and pH-responsive polymers bearing L-leucine residues in the side chains. Polymer (2000) 41:8247-8256.

Biomimetic responsive polymers with chiral side chains.

58. KOST J, HORBETT TA, RATNER BD, SINGH M: Glucose sensitive membranes containing glucose oxidase: activity, swelling and permeability studies. J. Biomed Mater. Res. 1985 19:1117-1133.

59. KOST J, LANGER R: Responsive polymer systems for controlled delivery of therapeutics. Trends Biotechnol. (1992) 10:127-131.

60. GOLDRAICH M, KOST J: Glucose-sensitive polymeric matrices for controlled drug delivery. Clin. Mater. (1993) 13:135-142.

61. TRAITEL T, COHEN Y, KOST J: Characterisation of glucose-sensitive insulin release systems in simulated in vivo conditions. Biomaterials (2000) 21:1679-1687.

A detailed publication concerning the factors affecting insulin release from polymer gels containing immobilised glucose oxidase and catalase.



62. PODUAL K, DOYLE III FJ, PEPPAS NA: Preparation and dynamic response of cationic copolymer hydrogels containing glucose oxidase. Polymer (2000) 41:3975-3983.

63. Polyethylene Glycol Chemistry. Harris JM (Ed.), Plenum Press, New York, USA (1992).

64. IMANISHI Y, ITO Y: Glucose-sensitive insulin-releasing molecular systems. Pure Appl. Chem. (1995) 67:2015-2021.

65. LEE SJ, PARK K: Synthesis and characterization of sol-gel phase-reversible hydrogels sensitive to glucose. J. Mol. Recogn. (1997) 9:549-557.

66. OBAIDAT AA, PARK K: Characterization of protein release through glucose-sensitive hydrogel membranes. Biomaterials (1997) 18:801-806.

67. AOKI T, NISHIMURA T, SANUI K, OGATA N: Phase-transition changes of poly((N-(S)-sec-butlyacrylamide-co-N-isopropylacrylamide) in response to amino acids and its chiral recognition. React. Func. Polym. (1998) 37:299-303.

68. FUJIKI M: Helix magic. Thermo-driven chiroptical switching and screw-sense inversion of flexible rod helical polysilylenes. J. Am. Chem. Soc. (2000) 122:3336-3343.

69. YASHIMA E, MAEDA Y, OKAMOTO Y: Synthesis of poly[N-(4-ethynylbenzyl)ephedrine] and its use as a polymeric catalyst for enantioselective addition of dialkylzincs to benzaldehyde. Polym. J. (1999) 31(Part 2):1033-1036.

70. YASHIMA E, MAEDA Y, OKAMOTO Y: Helix-helix transition of optically active poly((1R,2S)-N-(4-ethynylbenzyl)norephedrine) induced by diastereomeric acid-base complexation using chiral stimuli. J. Am. Chem. Soc. (1998) 120:8895-8896.

71. PARK K, ROBINSON JR: Bioadhesive polymers as platforms for oral controlled drug delivery: method to study bioadhesion. Int. J. Pharm. (1984) 19:107-127.

An early publication describing bioadhesive polymers.

72. SMART JD, KELLAWAY IW, WORTHINGTON HEC: An in vitro investigation of mucosa-adhesive materials for use in controlled drug delivery. J. Pharm. Pharmacol. (1984) 36:295-299.

One of the first examples employing mucoadhesion as a method for controlled drug delivery.

73. LEHR C-M: From sticky stuff to sweet

receptors - achievement, limits and novel approaches to bioadhesion. Eur. J. Drug Metab. Pharmacokinet. (1996) 21:397-405.

A review considering the mechanisms of lectin-sugar interactions and their role as bioadhesive formulations.

74. PEPPAS NA, SAHLIN JJ: Hydrogels as mucoadhesive and bioadhesive materials: a review. Biomaterials (1996) 17:1553-1561.

A useful overview of the different types of hydrogel being developed for drug delivery, and discussion of the mechanisms of bioadhesive-enhancement of drug targeting.

75. HUANG YB, LEOBANDUNG W, FOSS A et al.: Molecular aspects of muco- and bioadhesion: Tethered structures and site-specific surfaces. J. Control Rel. (2000) 65:63-71.

76. LEHR C-M: Lectin-mediated drug delivery. A second generation of bioadhesives. J. Control. Rel. (2000) 65:19-29.

A paper describing cytoadhesive polymers as novel drug delivery vehicles, with particular emphasis on internalisation pathways of liposome-lectin conjugates.

77. EASSON JH, HALTNER E, LEHR C-M, JAHN D: Bacterial invasin factors and lectins as second generation bioadhesives. In: Bioadhesive Drug Delivery Systems - Fundamentals, Novel Approaches and Developments. Mathiowitz E, Chickering DE, Lehr C-M (Eds.), Marcel Dekker, New York, USA (1999):409-432.

78. DAVIS BG: Recent developments in glycoconjugates. J. Chem. Soc. Perkin Trans. 1 (1999):3215-3237.

A thorough review of the leading developments in synthetic glycoscience, detailing syntheses of sugar-containing polymers, glycoproteins and potential applications.

79. SHARON N, LIS H: Lectins - proteins with a sweet tooth: functions in cell recognition. Essays Biochem. (1995) 30:59-75.

80. MAMMEN M, CHOI SK, WHITESIDES GM: Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem. Int. Ed. Engl. (1998) 37(20):2755-2794.

An important account of the mechanisms of molecular recognition in biology and the preparation of synthetic receptors for mediating biorecognition.

81. CHOI SK, MAMMEN M, WHITESIDES GM: Generation and in situ evaluation of

libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination. J. Am. Chem. Soc. (1997) 119(18):4103-4111.

The synthesis of anti-influenza glycopolymers by an elegant and facile pseudo-combinatorial route.

82. CHOI SK, LEE S, WHITESIDES GM: Synthesis of C-5 analogs of N-acetylneuraminic acid via indium-mediated allylation of N-substituted 2-amino-2-deoxymannoses. J. Org. Chem. (1996) 61(25):8739-8745.

83. GRECKI D: Gene therapy: prospects and problems. Emerging Drugs (1999) 4:247-261.

Review of the principal goals in gene therapy and possible obstacles to effective gene-based therapeutics.

84. KABANOV AV, KABANOV VA: DNA complexes with polycations for the delivery of genetic material into cells. Bioconj. Chem. (1995) 6:7-20.

85. FELGNER JH, KUMAR R, SRIDHAR CN et al.: Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. (1994) 269:2550-2561.

86. WANG CY, HUANG L: Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes. Biochemistry (1989) 28:9508 -9514.

Early example of the use of liposomes in DNA transfection.

87. OUPICK D, KONK C, ULBRICH K et al.: DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers. J. Control. Rel. (2000) 65:149-171.

A very detailed account of the synthesis and gene therapy applications of novel cationic copolymers.

88. VAN DE WETERING P, CHERNG J-Y, TALSMA H, CROMMELIN DJA, HENNINK WE: 2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents. J. Control. Rel. (1998) 53:145-153.

89. POUTON CW, SEYMOUR LW: Key issues in non-viral gene delivery. Adv. Drug Deliver. Rev. (2001) 46:187-203.

A comprehensive review of potential gene therapies.

90. ZABNER J, FASBENDER AI, MONINGER T, POELLINGER KA, WELSH MJ: Cellular and molecular barriers to gene transfer by a cationic lipid.

Alexander


J. Biol. Chem. (1995) 270:18997-19007.

91. XU Y, SZOKA F, JR.: Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry (1996) 35:5616-5623.

92. CAMILLERI P, KREMER A, EDWARDS AJ et al.: A novel class of cationic gemini surfactants showing efficient in vitro gene transfection properties. Chem. Commun. (2000):1253-1254.

93. POUTON CW, LUCAS P, THOMAS BJ et al.: Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. J. Control. Rel. (1998) 53:289-299.

94. THOMAS J, TIRRELL DA: Polyelectrolyte-sensitized phospholipid vesicles. Acc. Chem. Res. (1992) 25:336-342.

95. TOSTESON MT, HOLMES SJ, RAZIN M, TOSTESON DC: Mellitin lysis of red blood cells. J. Membrane Biol. (1985) 87:35-44.

96. STAYTON PS, HOFFMAN AS, MURTHY N et al.: Molecular engineering of proteins and polymers for targeting and intracellul

synthetic polymer systems in drug delivery

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