j lipos res bacon a, caparroswanderley w, zadi � and gregoriadis g induction of a cytotoxic t lymphocyte ctl response to plasmid dna delivered by lipodine j lipos res johnson pa, conwey ma, daly j, nicolson c, robertson j and mills kh plasmid dna encoding influenza virus haemagglutinin induces th cells and protection against respiratory infection despite its limited ability to generate antibody responses} gen virol sha z, vincent mj and compans rw title immunobiology gursel m, tunca s, ozkan m, ozcengiz g and alaeddinoglu g immunoadjuvant action of plasmid dna in liposomes vaccine this page is intentionally left blank polymer micelles as drug carriers elena v batrakova, tatiana k bronich, joseph a vetro and alexander v kabanov introduction it has long been recognized that improving one or more of the intrinsic adsorption, distribution, metabolism, and excretion adme properties of a drug is a critical step in developing more effective drug therapies as early as , paul ehrlich proposed altering drug distribution by conjugating toxic drugs to magic bullets antibodies having high affinity for cancer cellspecific antigens, in order to both improve the therapeutic efficacy of cancer while decreasing nifedipine breast milk its toxicity since then, it has become clear that directly improving intrinsic adme through modifications of the drug is limited or precluded by structural requirements for activity in other words, low molecular mass drugs are too small and have only limited number of atomic groups that can be altered to improve adme, which often adversely affects drug pharmacological activity in turn, the modifications of many low molecular mass drugs, aimed to increase their pharmacological activity, often adversely affect their adme properties for example, the potency and specificity of drugs can be improved by the addition of hydrophobic groups the associated decrease in water solubility, however, increases the likelihood of drug aggregation, leading to poor absorption and bioavailability during oral administration or lowered systemic bioavailability, high local toxicity, and possible pulmonary embolism during intravenous administration although there have been considerable difficulties for improving some existing drugs through chemical modifications, the problem became even more obvious with the development of highthroughput drug discovery technologies almost half of lead drug candidates identified by highthroughput screening have poor solubility in water, and are abandoned before the formulation development stage in addition, newly synthesized nifedipine breast milk drug candidates often fail due to poor bioavailability, metabolism andor undesirable side effects, which together decrease the therapeutic index of the molecules furthermore, a new generation of biopharma ceuticals and gene therapy agents are emerging based on novel biomacromolecules, such as dna and proteins the use of these biotechnologyderived drugs is completely dependent on efficient delivery to the critical site of the action in the body therefore, drug delivery research is essential in the translation of newly discovered molecules into potent drug candidates and can significantly improve therapies of existing drugs polymerbased drugs and drug delivery systems emerged from the laboratory bench in the s as a promising therapeutic strategy for the treatment of certain devastating human diseases, a number of polymer therapeutics are presently on the market or undergoing clinical evaluation to treat cancer and other diseases most of them are low molecular weight drug molecules or therapeutic proteins that are chemically linked to watersoluble polymers to increase drug solubility, drug stability, or enable targeting to tumors recently, as a result of rapid development of novel nanotechnologyderived materials, a new generation of polymer therapeutics has emerged, using materials nifedipine breast milk and devices of nanoscale size for the delivery of drugs, genes, and imaging molecules these materials include polymer micelles, polymerdn a complexes polyplexes, liposomes, and other nanostructured materials for medical use that are collectively known as nanomedicines compared with first generation polymer therapeutics, the new generation nanomedicines are more advanced they entrap small drugs or biopharmaceutical agents such as therapeutic proteins and dna, and can be designed to trigger the release of these agents at the target site many nanomedicines are constructed using selfassembly principles such as the spontaneous formation of micelles or interpolyelectrolyte complexes, driven by diverse molecular interactions hydrophobic, electrostatic, etc this chapter considers polymeric micelles as an important example of the new generation of nanomedicines, which is also perhaps among the most advanced approach toward clinical applications in diagnostics and the treatment of human diseases polymer micelle structures selfassembled micelles selfassembled polymer micelles are created from amphiphilic polymers that spontaneously form nanosized aggregates when the individual polymer chains polymeric micelle single polymer chains unimers hydrophobic core [unimer] cmc temp cmt [unimer] cmc temp cmt hydrophilic shell hydrophobic block hydrophilic block fig selfassembly of block copolymer micelles nifedipine breast milk unimers are directly dissolved in aqueous solution dissolution method above a threshold concentration critical micelle concentration or cmc and solution temperature critical micelle temperature or cmt fig amphiphilic polymers with very low water solubility can alternatively be dissolved in a volatile organic solvent, then dialyzed against an aqueous buffer dialysis method amphiphilic diblock hydrophilichydrophobic or triblock hydrophilic hydrophobichydrophilic copolymers are most commonly used to prepare self assembled polymer micelles for drug delivery although the use of graft copolymers has been reported for drug delivery purposes, the individual unimers are designed to be biodegradable, andor have a low enough molecular mass kda to be eliminated by renal clearance, in order to avoid polymer buildup within the body that can potentially lead to toxicity the most developed amphiphilic block copolymers assemble into spherical coreshell micelles approximately to nm in diameter, consisting of a hydrophobic core for drug loading and a hydrophilic shell that acts as a physical steric barrier to both micelle aggregation in solution, and to protein binding and opsonization during systemic administration fig the most common hydrophilic block used to form the hydrophilic shell is the fdaapproved excipient polyethylene nifedipine breast milk glycol peg or polyethylene oxide peo peg or peo consists of the same repeating monomer subunit chcho, and may have different terminal end groups, depending on the synthesis procedure, eg hydroxyl group �������� methoxy group chchchnh, etc pegpeo blocks typically range from to kda in addition to its fda approval, peg is extremely soluble and has a large excluded volume this makes it especially suitable for physically interfering with intramicelle interactions and subsequent micelle aggregation peg also blocks protein and cell surface interactions, which greatly decreases nanoparticle uptake by the reticuloendothelial system res, and consequently increases the plasma selfassembled no self assembly diblock copolymer ������� triblock copolymer njtj homopolymer dendritic covalentlvassembled unimolecular micelles x star hydrophilic block[jff hydrophobic block cationic block graft copolymer charged copolymer n n � � anionic block fig polymer micelle structures half life of the polymer micelle the degree of steric protection by the hydrophilic shell is a function of both the density and length of the hydrophilic peg blocks unlike the hydrophilic block, which is typically peg or peo, different types of hydrophobic blocks have been sufficiently developed as hydrophobic drug loading cores examples nifedipine breast milk of diblock copolymers include a polylamino acids, b biodegradable polyesters, which includes polyglycolic acid, polyd lactic acid, polyd,llactic acid, copolymers of lactideglycolide, and polye caprolactone, c phospholipidslong chain fatty acids and for triblock copolymers, d polypropylene oxide in pluronicspoloxamers the choice of hydrophobic block is largely dictated by drug compatibility with the hydrophobic core when drug is physically loaded, as described later and the kinetic stability of the micelle the selfassembly of amphiphilic copolymers is a thermodynamic and, consequently, a reversible process that is entropically driven by the release of ordered water from hydrophobic blocks it is either stabilized or destabilized by solvent interactions with the hydrophilic shell as such, the structural potential of amphiphilic copolymer unimers to form micelles is determined by the mass ratio of hydrophilic to hydrophobic blocks, which also affects the subsequent morphology if aggregates are formed if the mass of the hydrophilic block is too great, the copolymers exist in aqueous solution as unimers, whereas, if the mass of the hydrophobic block is too great, unimer aggregates with nonmicellar morphology are formed if the mass of the hydrophilic block is similar or slightly nifedipine breast milk greater than the hydrophobic block, then conventional core shell micelles are formed an important consideration for drug delivery is the relative thermodynamic potential for disassembly and kinetic rate of disassembly stability of the polymer micelle complexes, after intravenous injection and subsequent extreme dilution in the vascular compartment this is because the polymer micelles must be stable enough to avoid burst release of the drug cargo, as in the case of a physically loaded drug, upon systemic administration and remain as nanoparticles long enough to accumulate in sufficient concentrations at the target site the relative thermodynamic stability of polymer micelles which is inversely related to the cmc is primarily controled by the length of the hydrophobic block an increase in the length of the hydrophobic block alone significantly decreases the cmc of the unimer construct ie increases the thermodynamic stability of the polymer micelle, whereas an increase in the hydrophilic block alone slightly increases the cmc ie decrease the thermodynamic stability of a polymer micelle although the cmc indicates the unimer concentration below which polymer micelles will begin to disassemble, the kinetic stability determines the rate at which polymer micelle nifedipine breast milk disassembly occurs many diblock copolymer micelles possess good kinetic stability and only slowly dissociate into unimers after extreme dilution thus, although polymer micelles are diluted well below typical unimer cmcs m after intravenous injection, their relative kinetic stability might still be suitable for drug delivery the kinetic stability depends on several factors, including the size of a hydrophobic block, the mass ratio of hydrophilic to hydrophobic blocks, and the physical state of the micelle core the incorporation of hydrophobic drugs may also further enhance micelle stability unimolecular micelles unimolecular micelles are topologically similar to selfassembled micelles, but consist of single polymer molecules with covalently linked amphiphile chains for example, copolymers with starlike or dendritic architecture, depending on their structure and composition, can either aggregate into multimolecular micelles, or exist as unimolecular micelles dendrimers are widely used as building blocks to prepare unimolecular micelles, because they are highlybranched, have well defined globular shape and controled surface functionality for example, unimolecular micelles were prepared by coupling dendritic hypercores of different generations with peo chains the dendritic cores can entrap various drug molecules however, due to the structural limitations involved in the nifedipine breast milk synthesis of dendrimers of higher generation, and relatively compact structure of the dendrimers, the loading capacity of such micelles is limited thus, to increase the loading capacity, the dendrimer core can be modified with hydrophobic block, followed by the attachment of the peo chains for example, wang et al recently synthesized an amphiphilic arm star polymer with a polyamidoamine dendrimer core and arms composed of inner lipophilic polyecaprolactone block and outer peo block these unimolecular micelles were shown to encapsulate a hydrophobic drug, etoposide, with high loading capacity multiarm starlike block copolymers represent another type of unimolecular micelles star polymers are generally synthesized by either the armfirst or corefirst methods in the armfirst method, monofunctional living linear macromolecules are synthesized and then crosslinked either through propagation, using a bifunctional comonomer, or by adding a multifunctional terminating agent to connect precise number of arms to one center conversely, in the corefirst method, polymer chains are grown from a multifunctional initiator one of the first reported examples of unimolecular micelles, suitable for drug delivery, was a threearm star polymer, composed of mucic acid substituted with fatty acids as a lipophilic inner block nifedipine breast milk and peo as a hydrophilic outer block these polymers were directly dispersible in aqueous solutions and formed unimolecular micelles the size and solubilizing capacity of the micelles were varied by changing the ratio of the hydrophilic and lipophilic moieties in addition, starcopolymers with poly electrolyte arms can be prepared to develop phsensitive unimolecular micelles as drug carriers crosslinked micelles the multimolecular micelles structure can be reinforced by the formation of crosslinks between the polymer chains these resulting crosslinked micelles are, in essence, single molecules of nanoscale size that are stabile upon dilution, shear forces and environmental variations eg changes in ph, ionic strength, solvents etc there are several reports on the stabilization of the polymer micelles by cross linking either within the core domain or throughout the shell layer in these cases, the crosslinked micelles maintained small size and coreshell morphology, while their dissociation was permanently suppressed stable nanospheres from the peobpolylactide micelles were prepared by using polymerizable group at the core segment in addition to stabilization, the core polymerized micelles readily solubilized rather large molecules such as paclitaxel, and retained high loading capacity even upon dilution formation of nifedipine breast milk interpenetrating network of a temperaturesensitive polymer polynisopropylacrilomide inside the core was also employed for the stabilization of the pluronic micelles the resulting micelle structures were stable against dilution, exhibited temperatureresponsive swelling behavior, and showed higher drug loading capacity than regular pluronic micelles recently, a novel type of polymer micelles with crosslinked ionic cores was prepared by using block ionomer complexes as templates the nanofabrication of these micelles involved condensation of peobpolysodium methacrylate diblock copolymers by divalent metal cations into spherical micelles of coreshell morphology the core of the micelle was further chemically crosslinked and cations removed by dialysis resulting micelles represent hydrophilic nanospheres of core shell morphology the core comprises a network of the crosslinked polyanions and can encapsulate oppositely charged therapeutic and diagnostic agents, while a hydrophilic peo shell provides for increased solubility furthermore, these micelles displayed the ph and ionic strengthresponsive hydrogellike behavior, due to the effect of the crosslinked ionic core such behavior is instrumental for the design of drug carriers with controled loading and release characteristics drug loading and release in general, there are three major methods for loading drugs into polymer micelle cores chemical nifedipine breast milk conjugation, physical entrapment or solubilization, and polyionic complexation eg ionic binding chemical conjugation drug incorporation into polymer micelles via chemical conjugation was first proposed by ringsdorfs group in according to this approach, a drug is chemically conjugated to the coreforming block of the copolymer via a carefully designed ph or enzymesensitive linker, that can be cleaved to release a drug in its active form within a cell, the polymerdrug conjugate then acts as a polymer prodrug which self assembles into a coreshell structure the appropriate choice of conjugating bond depends on specific applications the nature of the polymerdrug linkage and the stability of the drug conjugate linkage can be controled to influence the rate of drug release, and therefore, the effectiveness of the prodrug for instance, recent work by kataokas group proposed phsensitive polymer micelles of peobpolyaspartate hydrazone doxorubicin, in which doxorubicin was conjugated to the hydrophobic segments through acidsensitive hydrazone linkers that are stable at extracellular ph , but degrade and release the free drug at acidic ph to in endosomes and lysosomes, the original approach developed by this group used doxorubicin conjugated to the polyaspartic acid chain of nifedipine breast milk peobpolyaspartic acid block copolymer through an amide bond adjusting both the composition of the block copolymer and the concentration of the conjugated doxorubicin, led to improved efficacy, as evidenced by a complete elimination of solid tumors implanted in mice it was later determined that doxorubicin physically encapsulated within the micellar core was responsible for antitumor activity this finding led to the use of peobpolyaspartate doxorubicin conjugates as nanocontainers for physically entrapped doxorubicin physical entrapment the physical incorporation or solublization of drugs within block copolymer micelles is generally preferred over micelleforming polymerdrug conjugates, especially for hydrophobic drug molecules indeed, many polymers and drug molecules do not contain reactive functional groups for chemical conjugation, and therefore, specific block copolymers have to be designed for a given type of drug in contrast, a variety of drugs can be physically incorporated into the core of the micelles, by engineering the structure of the coreforming segment in rivastigmine intermediate addition, molecular characteristics ie molecular weight, composition, presence of functional groups for active targeting within a homologous copolymer series can be designed to optimize the performance of a drug for a given drug delivery situation this concept nifedipine breast milk was introduced by our group in the late s and was initially termed micellar microcontainer, but is now widely known as a micel lar nanocontainer haloperidol was encapsulated in pluronic block copolymer micelles, the micelles were targeted to the brain using brainspecific antibodies or insulin, and enhancement of neuroleptic activity by the solubilized drug was observed during the last years, a large variety of amphiphilic block copolymers have been explored as nanocontainers for various drugs different loading methods can be used for physical entrapment of the drug into the micelles, including but not limited to dialysis, oil in water emulsification, direct dissolution, or solvent evaporation techniques, depending on the method, drug solubilization may occur during or after micelle assembly the loading capacity of the polymer micelles, which is frequently expressed in terms of the micellewater partition coefficient, is influenced by several factors, including both the structure of coreforming block and a drug, molecular characteristics of the copolymer such as composition, molecular weight, and the solution temperature many studies indicate that the most important factor related to the drug solubilization capacity of a polymer micelle is the compatibility between the nifedipine breast milk drug and the coreforming block for this reason, the choice of the coreforming block is most critical one parameter that can be used to assess the compatibility between the polymer and a drug is the floryhuggins interaction parameter, xsp defined as xsp= ss $pvskt, where s and p are scatchardhildebrand solubility parameters, and vs is the molecular volume of the solubilizate it was successfully used as a correlation parameter for the solubilization of aliphatic and aromatic hydrocarbons in block copolymer micelles, recently, allens group elegantly demonstrated that the calculation and comparison of partial solubility parameters of polymers and drugs could be used as a reliable means to predict polymerdrug compatibility and to guide formulation development polymer micelles, possessing coreforming blocks predicted to be compatible with the drug of interest ellipticine, were able to increase the solubility of the drug up to , times, compared with its saturation solubility in water the degree of compatibility between the drug and the coreforming block has also been shown to influence the release rate of the drug from the micelles when the environment within the core of the micelle becomes more compatible with the nifedipine breast milk drug, it results in a considerable decrease in the rate of drug release for a given drug, the extent of incorporation is a function of factors that also control the micelle size andor aggregation number such factors include the ratio of hydrophobic to hydrophilic block length and the copolymer molecular weight for example, the loading capacity of pluronic micelles was found to increase with the increase in the hydrophobic ppo block length this effect is attributed to a decrease in cmc, and therefore, an increase in aggregation number and micelle core size also, but to a lesser extent, the hydrophilic block length affects the extent of solubilization, such that an increase in percentage of peo in pluronic block copolymers results in a decrease in the loading capacity of the micelles, for a given ratio of ppotopeo, higher molecular weight polymers form larger micelles, and therefore, show a higher drug loading capacity therefore, the total amount of loaded drug can be adjusted as a function of the micellar characteristics as clearly was demonstrated by nagaradjan and kozlov et al several studies indicate that both the copolymer concentration as well as nifedipine breast milk the drug to polymer ratio upon loading, have a complex effect on the loading capacity of polymer micelles in general, more polymer chains provide more absorption sites as a result, solubilization is increased with polymer concentration however, the solubilization capacity was found to reach a saturation level with an increase of polymer concentration the maximum loading level is largely influenced by the interaction between the solubilizate and coreforming block, and stronger interactions enable saturation to be reached at lower polymer concentration it was also demonstrated in the studies by hurter and hatton, that the loading capacity of micelles formed from copolymers with high hydrophobic content was independent of the polymer concentration in addition, the location of the incorporated molecules within polymer micelles micelle core or the coreshell interface determines the extent of solubilization, as well as the rate of drug release, it has been found that more soluble compounds are localized at the coreshell interface or even in the inner shell, whereas more hydrophobic molecules have a tendency to solubilize in the micelle core the release rate of drug localized in the shell or at the interface appears to nifedipine breast milk account for the burst release from the micelles in general, for drugs physically incorporated in polymer micelles, release is controled by the rate of diffusion of the drug from the micellar core, stability of the micelles, and the rate of biodegradation of the copolymer if the micelle is stable and the rate of polymer biodegradation is slow, the diffusion rate of the drug will be mainly determined by the abovementioned factors, ie the compatibility between the drug and core forming block of copolymer the amount of drug loaded, the molecular volume of drug, and the length of the core forming block in addition, the physical state of the micelle core and drug has a large influence on release characteristics it was demonstrated that the diffusion of incorporated molecules from the block copolymer micelles with glassy cores is slower, in comparison to the diffusion out of the cores that are more mobile poly ionic complexation charged therapeutic agents can be incorporated into block copolymer micelles, through electrostatic interactions with an oppositely charged ionic segment of block copolymer since it was being proposed independently by kabanov and kataoka in this approach nifedipine breast milk is now widely used for the incorporation of various polynucleic acids into block ionomer complexes, for developing nonviral gene delivery systems ionic block lengths, charge density, and ionic strength of the solution affect the formation of stable block ionomer complexes, and therefore, control the amount of drug that can be incorporated within the micelles, the ph and saltsensitivity of such block ionomer micelles provide a unique opportunity to control the triggered release of the active therapeutic agent furthermore, block ionomer complexes can participate in the polyion interchange reactions which are believed to account for the release of the therapeutic agent and dna in an active form inside cells several comprehensive reviews can be found in the literature that focus on block ionomer micelles as drug and gene delivery systems, in addition, physicochemical aspects of the dna complexes with cationic block copolymers have also been recently reviewed as an example, the metalcomplex formation of ionic block copolymer, peo bpolylaspartic acid, was explored to prepare polymer micelles incorporating czsdichlorodiamminoplatinum ii cddp, a potent chemotherapeutic agent widely used in the treatment of a variety of solid tumors, particularly, testicular, ovarian, head and neck, nifedipine breast milk and lung tumors, the cddploaded micelles had a size of approximately nm these micelles showed remarkable stability upon dilution in distilled water, while in physiological saline, they displayed sustained release of the regenerated pt complex over hrs, due to inverse ligand exchange from carboxylate to chloride the release rate was inversely correlated with the chain length of polylaspartic acid segments in the block copolymer the stability of cddploaded micelle against salt was shown to be improved by the addition of homopolymer, polylaspartic acid, in the micelles recently, cddploaded micelles were newly prepared using another block copolymer, peobpolyglutamic acid to improve and optimize the micellar stability, as well as the drug release profile the drug loading in the micelles was as high as ww, and these micelles released the platinum in physiological saline at �c in sustained manner hrs, without initial burst of the drug the principle of polyionic complexation can also be used to design new pho tosensitizers for photodynamic therapy of cancer the group of kataoka reported formation of micelles, as a result of mixing of oppositely charged dendrimer porphyrin and block ionomer, based on electrostatic assembly or nifedipine breast milk combination of electrostatic and hydrogen bonding interactions, the micelles were stabile at physiological conditions and released the entrapped dendrimers in the acidic ph environment ph , suggesting a possibility of phtriggered drug release in the intracellular endosomal compartments overall, the photodynamic efficacy of the dendrimer porphyrins was dramatically improved by inclusion into micelles this process resulted in more than two orders of magnitude increase in the photocyto toxicity, compared with that of the free dendrimer porphyrins in addition, the polyionic complexation has been used to immobilize charged enzymes such as egg white lysozyme or trypsin, which were incorporated in the core of polyion micelles, after mixing with oppositely charged ionic block copolymer a remarkable enhancement of enzymatic activity was observed in the core of the micelles furthermore, the onoff switching of the enzyme activity was achieved through the destabilization of the core domain by applying a pulse electric field these unique features of the polyion micelles are relevant for their use as smart nanoreactors in the diverse fields of medical and biological engineering last, but not the least, a special class of polyion complexes has been synthesized by reacting block nifedipine breast milk ionomers with surfactants of opposite charge, resulting in the formation of environmentally responsive nanomaterials, which differ in sizes and morphologies, and include micelles and vesicles these materials contain a hydrophobic core formed by the surfactant tail groups, and a hydrophilic shell formed, for example, by peo chains of the block ionomer these block ionomer complexes can incorporate charged surfactant drugs such as retinoic acid, as well as other drugs via solubilization in the hydrophobic domains formed by surfactant molecules they display transitions induced by changes in ph, salt concentration, chemical nature of low molecular mass counterions, as well as temperature they can also be fine tuned to respond to environmental changes occurring in a very wide range of conditions that could realize during delivery of biological and imaging agents, the unique selfassembly behavior, the simplicity of the preparation, and the wide variety of available surfactant components that can easily produce polymer micelles with a very broad range of core properties, make this type of materials extremely promising for developing vehicles for the delivery of diagnostic and therapeutic modalities pharmacokinetics and biodistribution incorporation of a low molecular mass drug into nifedipine breast milk polymer micelles drastically alters pharmacokinetics and biodistribution of the drug in the body, which is crucial for the drug action low molecular mass drugs, after administration in the body, rapidly extravasate to various tissues affecting them almost indiscriminately, and then are rapidly eliminated from the body via renal clearance, often causing toxicity to kidneys furthermore, many drugs display low stability and are degraded in the body, often forming toxic metabolites an example is doxorubicinol, a major metabolite of doxorubicin, which causes cardiac toxicity these impediments to the therapeutic use of low molecular mass drugs can be mitigated by encapsulating drugs in polymer micelles within the micelles, the drug molecules are protected from enzymatic degradation by the micelle shell the pharmacokinetics and biodistribution of the micelleincorporated drugs are mainly determined by the surface properties, size, and stability of the micelles, and are less affected by the properties of the loaded drug the surface properties of the micelles are determined by the micelle shell the shell from peo effectively masks drug molecules and prevents interactions with serum proteins and cells, which contributes to prolonged circulation of the micelles in the body nifedipine breast milk from the size standpoint, polymer micelles fit an ideal range of sizes for systemic drug delivery on the one hand, micelles are sufficiently large, usually exceeding nm in diameter, which hinders their extravasation in nontarget tissues and prevents renal glomerular excretion on the other hand, the micelles are not considered large, since their size usually does not exceed nm as a result, micelles avoid scavenging by the mononuclear phagocytes system mps in the liver and spleen to this end, stealth particles whose surface is decorated with peo are known to be less visible to macrophages and have prolonged halflives in the blood the contribution of the micelle stability to pharmacokintetics and biodistribution is much less understood, although it is clear that micelle degradation should result in a decrease of the size and drug release, perhaps, prematurely degradation of the micelles, resulting in the formation of block copolymer unimers, could also be a principal route for the removal of the polymer material from the body the molecular mass of the unimers of most block copolymers is below the renal excretion limit, ie less than to kda, while the molecular mass nifedipine breast milk of the micelles, which usually contain several dozen or even hundreds of unimers molecules, is above this limit thus, the unimers are sufficiently small and can be removed via renal excretion, while the micelles cannot a recent study by batrakova et al determined pharmacokinetic parameters of an amphiphilic block copolymer, pluronic p, and perhaps provided first evidence that the pharmacokinetic behavior of a block copolymer can be a function of its aggregation state specifically, the formation of micelles increased the halflife of the block copolymer in plasma and decreased the uptake of the block copolymer in the liver however, it had no effect on the total clearance, indicating that the elimination of pluronic p was controled by the renal tubular transport of unimers, but not by the rate of micelles disposition or disintegration furthermore, the values of the clearance suggested that a significant portion of the block copolymer was reabsorbed back into the blood, probably, through the kidneys tubular membranes chemical degradation of the polymers comprising the micelles, followed by renal excretion of the relatively low molecular mass products of degradation, may be another route for the removal of nifedipine breast milk the micelle polymer material from the body this route could be particularly important in the case of the crosslinked or unimolecular micelles, micelles displaying very high stability, andor micelles composed from very hydrophobic polymer molecules that can bind and retain considerably biological membranes and other cellular components the delivery of chemotherapeutic drugs to treat tumors is one of the most advanced areas of research using polymer micelles two approaches have been explored to enhance delivery of drugloaded polymer micelles to the tumor sites passive targeting and vectorized targeting the passive targeting involves enhanced permeability and retention epr effect it is based on the fact that solid tumors display increased vascular density and permeability caused by angio genesis, impaired lymphatic recovery, and lack of a smooth muscle layer in solid tumor vessels as a result, micellar drugs can penetrate and retain in the sites of tumor lesions at the same time, extravasation of micellar drugs in normal tissues is decreased, compared with low molecular drug molecules among normal organs, spleen and liver can accumulate polymer drugs, but the drugs are eventually cleared via the lymphatic system the increased circulation time nifedipine breast milk of the micellar drugs should further enhance exposure of the tumors to the micellar drug, compared with the low molecular mass drugs along with passive targeting, the delivery of micellar drugs to tumors can potentially be enhanced by the modification of the surface of the polymer micelles with the targeting molecules, vectors that can selectively bind to the surface of the tumor cells potential vectors include antibodies, aptamers and peptides, capable of binding tumorspecific antigens and other molecules diplayed at the surfaces of the tumors altered biodistribution of a common antineoplastic agent was demonstrated for cddp encapsulated in polyionic micelles with peobpolyglutamic acid block copolymers free cddp is rapidly distributed to each organ, where its levels peak at about one hr after iv administration in contrast, in the case of the cddp incorporated micelles, due to their remarkably prolonged blood circulation time, the drug level in the liver, spleen and tumor continued to increase up to at least hrs after injection consequently, the cddpincorporated micelle exhibited and fold higher accumulation in the liver, spleen and tumor respectively, than the free cddp at the same time, the encapsulation of cddp nifedipine breast milk into the micelles significantly decreased drug accumulation in the kidney, especially during first hr after administration this suggested potential for the decrease of severe nephrotoxicity observed with the free drug, which is excreted through the glomerular filtration, thus affecting the kidney promising results were also demonstrated for doxorubicin incorporated into styrenemaleic acid micelles in this case, as a result of drug entrapment into micelles, the drug was redirected from the heart to the tumor, and the doxorubicin cardiotoxicity was diminished complete blood counts and cardiac histology for the micellar drug showed no serious side effects for iv doses as high as mgkg doxorubicin equivalent in mice similar results were reported for doxorubicin incorporated in mixed micelles of peobpolylhistidine and peobpolyllactic acid block copolymers tissue levels of doxorubicin administered in the micellar formulation were decreased in the blood and the liver, and considerably increased in the solid tumor, compared with the free drug further increase in the tumor delivery was achieved by modifying the surface of the micelles with the folate molecules the accumulated doxorubicin levels observed using folatemodified micelles was times higher than those for free doxorubicin, and times nifedipine breast milk higher than those for unmodified micelles the first micellar formulation of doxorubicin to reach clinical evaluation stage, used the micelles composed of triblock copolymer, peobpoly propylene oxideb peo, pluronic analysis of pharmacokinetics and biodistribution of doxorubicin incorporated into mixed micelles of pluronics l and f, spc, demonstrated more efficient accumulation of the micellar drug in the tumors, compared with the free drug specifically, the areas under the curves auc in the lewis lung carcinoma ll m solid tumors in cb mice were increased about two fold using sp, compared with the free doxorubicin furthermore, this study indicated that the peak levels of doxorubicin formulated with sp in the tumor were delayed and the drug residence time was increased, in comparison with the free doxorubicin a clear visualization of drug delivery to the tumor site was shown for doxorubicin covalently incorporated through the phsensitive link into polymer micelles of peopolyaspartate hydrazone doxorubicin a phasecontrast image showed that the tumor blood vessels containing the micelles leaked into extra vascular compartments of the tumors, resulting in the infiltration of the micelles into tumor sites the micelles circulated in the blood for a prolonged nifedipine breast milk time, and the auc for micellar doxorubicin was fold greater than the auc for the free doxorubicin furthermore, the auc values of the micellar doxorubicin in the heart and kidney decreased, compared with the free drug thus, the selectivity of drug delivery to the tumor, compared with heart and kidney auctumoraucrgan was increased by and folds respectively this may result in the reduction of side effects of doxorubicin such as cardiotoxicity and nephrotoxicity moreover, the micellar doxorubicin showed relatively low uptake in the liver and spleen, despite very long residence time in the blood biodistribution of paclitaxel incorporated into biodegradable polymer micelles of monomethoxypeobpolyd,llactide block copolymer, genexolpm, was compared with the regular formulation of the drug in cremophor el two to threefold increases in drug levels were demonstrated in most tissues including liver, spleen, kidneys, lungs, heart and tumor, after iv administration of genexol pm, compared with paclitaxel nevertheless, acute dose toxicity of genexolpm was about times lower than that of the conventional drug formulation, which appears to be a result of the reformulation avoiding the use of chremophor el and dehydrated ethanol that are toxic selective tumor nifedipine breast milk targeting with paclitaxel encapsulated in micelles, modified with tumorspecific antibodies c immunomicelles, was reported using lewis lung carcinoma solid tumor model in cbj mice these micelles were prepared from peodistearyl phosphatidylethanolamine conjugates with the free peo end activated with the pnitrophenylcarbonyl group for the antibody attachment the amount of micellar drug accumulated in the tumor exceeded that in the non target tissue muscles by more than ten times it is worth noting that the highest accumulation in the tumor was demonstrated in the micelles containing the longest peo chains, which also had the longest circulation time in the blood furthermore, the immunomicelles displayed the highest amount of tumoraccumulated drug, compared with either free paclitaxel or nonvectorized micelles it was demonstrated that paclitaxel delivered by plain micelles in the interstitial space of the tumor was eventually cleared after gradual micellar degradation in contrast, paclitaxelloaded c immunomicelles were internalized by cancer cells and the retention of the drug inside the tumor was enhanced unexpected results were found using phsensitive polymer micelles of n isopropylacrylamide and methacrylic acid copolymers randomly or terminally alkylated with octadecyl groups it was demonstrated that aluminium chloride nifedipine breast milk phthalocyanine alclpc incorporated in such micelles was cleared more rapidly and less accumulated in the tumor, than the alclpc formulated with cremophor el furthermore, significant accumulation in the liver and spleen and lungs for most hydrophobic copolymers was observed, compared with cremophor el formulation the enhanced uptake of such polymer micelles by the cells of mononuclear phagocyte system mps could be due to micelle aggregation in the blood and embolism in the capillaries thus, it attempted to reduce the uptake of the micelles in mps by incorporating water soluble monomers, nvinylpyrrolidone in the copolymer structure the modified formulation displayed same levels of tumor accumulation and somewhat higher antitumor activity than the cremophor el formulation this work serves as an example reinforcing the need of proper adjustment of the polymer micelle structure, and perhaps the need of using block copolymers to produce a defined protective hydrophilic shell to facilitate evasion of the polymer micelles from mps drug delivery applications the studies on the application of polymer micelles in drug delivery have mostly focused on the following areas that are considered below delivery of anticancer agents to treat tumors drug delivery nifedipine breast milk to the brain to treat neurodegenerative diseases delivery of antifungal agents delivery of imaging agents for diagnostic applications and delivery of polynucleotide therapeutics chemotherapy of cancer to enhance chemotherapy of tumors using polymer micelles, four major approaches were employed passive targeting of polymer micelles to tumors due to epr effect targeting of polymer micelles to specific antigens overexpressed at the surface of tumor cells enhanced drug release at the tumor sites having low ph and sensitization of drug resistant tumors by block copolymers a series of pioneering studies by kataokas group used polymer micelles for passive targeting of various anticancer agents and chemotherapy of tumors, one notable recent example reported by this group involves polymer micelles of peobpolylaspartic acid incorporating cddp evaluation of anticancer activity using murine colon adenocarcinoma c as an in vivo tumor model, demonstrated that cddp in polymer micelles had significantly higher activity than the free cddp, resulting in complete eradication of the tumor a formulation of paclitaxel in biodegradable polymer micelles of monomethoxypeobpolyd,llactide block copolymer, genexolpm, also displayed elevated activity in vivo against human ovarian carcinoma ovcar and human breast carcinoma mcf, compared with nifedipine breast milk a regular formulation of the drug in cremophor el in addition, anthracy cline antibiotics, doxorubicin and pirarubicin, incorporated in styrenemaleic acid micelles each revealed potent anticancer effects in vivo against mouse sarcoma s, resulting in complete eradication of tumors in of tested animals notably, animals survived for more than one year, after treatment with the micelle incorporated pirarubicin at doses as high as loomgkg of pirarubicin equivalent complete blood counts, liver function test, and cardiac histology showed no sign of adverse effects for intravenous doses of the micellar formulation in contrast, animals receiving free pirarubicin had a much reduced survival and showed serious side effects collectively, these studies suggested that various micelle incorporated drugs display improved therapeutic index in solid tumors, which correlates with enhanced passive targeting of the drug to the tumor sites, as well as decreased side effects, compared with conventional formulations of these drugs tumorspecific targeting of polymer micelles to molecular markers expressed at the surface of the cancer cells has also been explored to eradicate tumor cells for example, a recent study by gaos group developed a polymer micelle carrier to deliver doxorubicin to the nifedipine breast milk tumor endothelial cells with overexpressed kvfb integrins a cyclic pentapeptide, crgd was used as a targeting ligand that is capable of selective and high affinity binding to the ��� integrin micelles of peo bpolyecaprolactone loaded with doxorubicin were covalently bound with crgd as a result of such modification, the uptake of doxorubicincontaining micelles in in vitro human endothelial cell model derived from kaposis sarcoma, was profoundly increased in addition, folate receptor often overexpressed in cancer cells has been evaluated for targeting various drug carriers to tumors this strategy has also been evaluated to target polymer micelles for example, mixed micelles of peobpolylhistidine and peobpolyllactic acid block copolymers with solubilized doxorubicin or micelles of peobpolydllacticcoglycolic acid block copolymer with covalently attached doxorubicin, were each surface modified by conjugating folate molecules to the free peo ends in both cases, in vitro and in vivo studies demonstrated increased antitumor activity of the micelle incorporated drug resulting from such modification the enhanced delivery of the micellar drugs through the folate receptor, and the enhanced retention of the modified micelles at the tumor sites are possible explanations for the effects of these folate modifications nifedipine breast milk micelles conjugated with antibodies or antibody fragments capable to recognize tumor antigens were shown to improve therapeutic efficacy in vivo over nonmodified micelles this approach can result in high selectivity of binding, internalization, and effective retention of the micelles in the tumor cells in addition, recent advances in antibody engineering allow for the production of humanized miconazole nitrate use and lipitor antibody fragments, reducing problems with immune response against mouse antibodies for example, micelles of peodistearyl phosphatidylethanolamine were covalently modified with the monoclonal antibody c that binds to nucle osomes, displayed at the surface of many tumor cells the micelles were then used for incorporating various poorly soluble anticancer drugs including tamoxifen, paclitaxel, dequalinium, and chlorine e trimethyl ester it was shown that paclitaxelloaded cimmunomicelles could specifically recognize a variety of tumor types the binding of these immunomicelles was observed for all cancer cell lines tested, ie murine lewis lung carcinoma, tlymphoma el, and human breast adenocarcinomas, bt and mcf moreover, paclitaxelloaded c immunomicelles demonstrated highest anticancer activity in lewis lung carcinoma tumor model in mice, compared with plain paclitaxelloaded micelles and the free drug the increased antitumor effect of immunomicelles in vivo correlated nifedipine breast milk with the enhanced retention of the drug delivered with the immunomicelles inside the tumor tumors often display low ph of interstitial fluid, which is mainly attributed to higher rates of aerobic and anaerobic glycolysis in cancer cells than in normal cells this phenomenon has been employed in the design of various phsensitive polymer micelle systems for the delivery of anticancer drugs to the tumors one approach consisted in the chemical conjugation of anticancer drugs to the block copolymers through ph sensitive cleavable links that are stable at neutral ph, but are cleavable and release the drug in the mildly acidic ph for example, several groups used hydrasonebased linking groups, to covalently attach doxorubicin to peobpolydllacticcoglycolic acid block copolymer peo bblockpolyallyl glycidyl ether or peobpolyaspartate hydrazone block copolymer, it was suggested that doxorubicin will remain in the micelles in the blood stream, and will be released at tumor sites at lower ph for example, in vitro and in vivo studies using peobpolyaspartate hydrazone doxorubicin micelles demonstrated that the micelles display an intracellular phtriggered drug release capability, tumorinfiltrating permeability, and effective antitumor activity with extremely low toxicity, overall, the animal studies nifedipine breast milk suggested that such polymer micelle drug has a wide therapeutic window due to increased efficacy and decreased toxicity, compared with free doxorubicin an alternative mechanism for phinduced triggering of drug release at the tumor sites consists of using ph sensitive polyacids or polybases as building blocks for polymer micelles for example, mixed micelles of peob polylhistidine and peobpolyllactic acid block copolymers incorporate ph sensitive polybase, polylhistidine in the hydrophobic core the core can also solubilize hydrophobic drugs such as doxorubicin the protonation of the poly base at acidic conditions resulted in the destabilization of the core and triggered release of the drug this system was also targeted to the tumors through the folate molecules as described earlier and has shown significant in vivo antitumor activity and less side effects, compared with the free drug notably, it was also effective in vitro and in vivo against multidrug resistant mdr human breast carcinoma mcf adr that overexpresses pglycoprotein pgp pgp is a drug efflux transport protein that serves to eliminate drugs from the cancer cells and significantly decreases the anticancer activity of the drugs the micelle incorporated drug was released inside nifedipine breast milk the cells, and thus avoided the contact with pgp localized at the cell plasma membrane, which perhaps contributed to the increased activity of ph sensitive doxorubicin micelles in the mdr cells a different approach using pluronic block copolymer micelles to overcome mdr in tumors has been developed by our group, studies by alakhov et al demonstrated that pluronic block copolymers can sensitize mdr cells, resulting in an increased cytotoxic activity of doxorubicin, paclitaxel, and other drugs by , orders of magnitude remarkably, pluronic can enhance drug effects in mdr cells through multiple effects including inhibiting drug efflux transporters, such as pgp and multidrug resistance proteins mrps abolishing drug sequestration within cytoplasmic vesicles, inhibiting the glutathioneglutathione stransferase detoxification system, and enhancing proapoptotic signaling in mdr cells similar effects of pluronics have also been reported using in vivo tumor models, in these studies, mice bearing drugsensitive and drugresistant tumors were treated with doxorubicin alone and with doxorubicin in pluronic compositions the tumor panel included ip murine leukemias p, pdox, sc murine myelomas sp, spdnr, iv and sc lewis lung carcinoma llm, sc human breast carcinomas mcf, mcfadr, and sc human oral epidermoid carcinoma nifedipine breast milk kbv using the nci criteria for tumor inhibition and increased lifespan, pluronic doxorubicin has met the efficiency criteria in all models of , while doxorubicin alone was only effective in selected tumors of results showed that the tumors were more responsive in the pluronicdoxorubicin treatment groups than in doxorubicin alone these studies demonstrated improved treatment of drug resistant cancers with pluronics the mechanisms of effects of pluronic on pgp have been studied in great detail in particular, exposure of mdr cells to pluronics has resulted in the inhibition of pgpmediated efflux, and this overcomes defects in intracellular accumulation of pgpdependent drugs, and abolishes the directionality difference in the flux of these drugs across polarized cell monolayers the lack of changes in membrane permeability with pluronics to nonpgp compounds in mdr cells and pgp probes in nonmdr cells suggested that pluronic effects were specific to the pgp efflux system these effects were observed at pluronic concentrations less than or equal to the critical micelle concentration cmc, thus, pluronic unimers rather than the micelles were responsible for these effects specifically, pluronic molecules displayed a dual function in mdr cells firstly, they incorporated nifedipine breast milk into the cell membranes and decreased the membrane microviscosity this was accompanied by the inhibition of pgp atpase activity secondly, they translocated into cells and reached intracellular compartments this was accompanied by the inhibition of respiration, presumably due to pluronic interactions with the mitochondria membranes as a result, within min after exposure to select pluronics, intracellular levels of atp in mdr cells were drastically decreased remarkably, such atp depletion was not observed in nonmdr cells, suggesting that the pluronic was selective, with respect to the mdr phenotype, combining these two effects, pgp atpase inhibition and atp depletion, resulted in the shutdown of the efflux system in mdr cells the pgp remained functionally active when atp was restored using an atp supplementation system in the presence of a pluronic, or when atp was depleted, but there was no direct contact between the pluronic and pgp and no atpase inhibition overall, these detailed studies which resulted in the development of a micellar formulation of doxorubicin that is evaluated clinically, reinforce the fact that block copolymers, comprising the micelles, can serve as biological response modifying agents that can have beneficial effects in nifedipine breast milk the chemotherapy of tumors drug delivery to the brain by restricting drug transport to the brain, the blood brain barrier bbb represents a formidable impediment for the treatment of brain tumors and neurodegenerative diseases such as hivassociated dementia, stroke, parkinsons and alzheimers diseases two strategies using polymer micelles have been evaluated to enhance delivery of biologically active agents to the brain the first strategy is based on the modification of polymer micelles with antibodies or ligand molecules capable of transcytosis across brain microvessel endothelial cells, comprising the bbb the second strategy uses pluronic block copolymers to inhibit drug efflux systems, particularly, pgp, and selectively increase the permeability of bbb to pgp substrates an earlier study used micelles of pluronic block copolymers for the delivery of the cns drugs to the brain, these micelles were surfacemodified by attaching to the free peo ends, either polyclonal antibodies against brainspecific antigen, glycoprotein, or insulin to target the receptor at the lumenal side of bbb the modified micelles were used to solubilize fluorescent dye or neuroleptic drug, haloperidol, and these formulations were administered intravenously in mice both the antibody and insulin modification of nifedipine breast milk the micelles resulted in enhanced delivery of the fluorescent dye to the brain and drastic increases in neuroleptic effect of haloperidol in the animals subsequent studies using in vitro bbb models demonstrated that the micelles, vectorized by insulin, undergo receptormediated transport across brain microvessel endothelial cells based on one of these observations, one should expect development of novel polymer micelles that target specific receptors at the surface of the bbb to enhance transport of the incorporated drugs to the brain the studies by our group have also demonstrated that selected pluronic block copolymers, such as pluronic p, are potent inhibitors of pgp, and they have the increased entry of the pgpsubstrates to the brain across bbb pluronic did not induce toxic effect in bbb, as revealed by the lack of alteration in paracellular permeability of the barrier, and in histological studies, using specific markers for brain endothelial cells overall, this strategy has potential in developing novel modalities for the delivery of various drugs to the brain, including selective anti cancer agents to treat metastatic brain tumors, as well as hiv protease inhibitors to eradicate hiv virus in the brain nifedipine breast milk formulations of antifungal agents the need for safe and effective modalities for the delivery of chemotherapeutic agents to treat systemic fungal infections in immunocompromised aids, surgery, transplant and cancer patients is very high the challenges to the delivery of antifungal agents include low solubility and sometimes high toxicity of these agents these agents, such as amphotericin b, have low compatibility with hydrophobic cores of polymer micelles formed by many conventional block copolymers thus, to increase solubilization of amphotericin b, the coreforming blocks of methoxypeo bpolylaspartate were derivatized with stearate side chains the resulting block copolymers formed micelles amphotericin � interacted strongly with the stearate side chains in the core of the micelles, resulting in an efficient entrapment of the drug in the micelles, as well as subsequent sustained release in the external environment as a result of solubilization of amphotericin � in the micelles, the onset of hemolytic activity of this drug toward bovine erythrocytes was delayed, relative to that of the free drug using a neutropenic murine model of disseminated candidas, it was shown that micelleincorporated amphotericin � retained potent in vivo activity pluronic block copolymers were used nifedipine breast milk by the same group for incapsulation of another poorly soluble antifungal agent, nystatin this is a commercially available drug that has shown potential for systemic administration, but has never been approved for that purpose, due to toxicity issues the possibility to use pluronic block copolymers to overcome resistance to certain antifungal agents has also been demonstrated overall, one should expect further scientific developments using polymer micelle delivery systems for the treatment of fungal infection delivery of imaging agents efficient delivery of imaging agents to the site of disease in the body can improve early diagnostics of cancer and other diseases the studies in this area using polymer micelles as carriers for imaging agents were initiated by torchilin for example, micelles of amphiphilic peolipid conjugates were loaded with in and gadolinium diethylenetriamine pentaacetic acidphosphatidylethanolamine gd dtpape and then used for visualization of local lymphatic chain after subcutaneous injection into the rabbits paw the images of local lymphatics were acquired using a gamma camera and a magnetic resonance mr imager the injected micelles stayed within the lymph fluid, thus serving as lymphangiographic agents for indirect mr or gamma lymphography another polymer nifedipine breast milk micelle system composed of amphiphilic methoxypeobpoly[epsilon,ntriiodobenzoyll lysine] block copolymers, labeled with iodine, was administered systemically in rabbits and visualized by xray computed tomography the labeled micelles displayed exceptional hrs halflife in the blood, which is likely due to the core shell architecture of the micelle carriers that protected the iodinecontaining core notably, small polymer micelles nm may be advantageous for bioimaging of tumors, compared with pegmodified longcirculating liposomes ca loonm in particular, the micelles from peodistearoyl phosphatidyl ethanolamine conjugates containing inlabeled model protein were more efficacious in the delivery of protein to lewis lung carcinoma than larger longcirculating liposomes overall, polymer micelles loaded with various agents for gamma, magnetic resonance, and computed tomography imaging represent promising modalities for noninvasive diagnostics of various diseases delivery of polynucleo tides to improve the stability of polycationbased dna, delivery complexes in dispersion block and graft copolymers containing segments from polycations and non ionic watersoluble polymers, such as peo, were developed binding of these copolymers with dna results in the formation of micellelike block ionomer complexes polyion complex micelles, containing hydrophobic sites formed by the polycationneutralized dna and hydrophilic sites formed by the nifedipine breast milk peo chains despite neutralization of charge, complexes remain stable in aqueous dispersion due to the effect of the peo chains overall, the peo modified polycationdna complexes form stable dispersions and do not interact with serum proteins, these systems were successfully used for intravitreal delivery of an antisense oligonucleotide and the suppression of gene expression in retina in rats furthermore, they displayed extended plasma clearance kinetics and were shown to transfect liver and tumor cells, after systemic administration in the body in addition, there is a possibility targeting such polyplexes to the specific receptors at the surface of the cell, for example, by modifying the free ends of peo chains with specific targeting ligands alternatively, to increase the binding of the complexes with the cell membrane and the transport of the polynucleotides inside the cells, the polycations were modified with amphiphilic pluronic molecules, one recent study has shown a potential of pluronicpolyethyleneiminebased micelles for in vivo delivery of antisense oligonucleotides to tumors, and have demonstrated sensitization of the tumors to radiotherapy as a result of systemic administration of the oligonucleotideloaded micelles clinical trials three polymer micelle formulations of anticancer drugs nifedipine breast milk have been reported to reach clinical trials the doxorubicinconjugated polymer micelles developed by kataokas group have progressed recently to phase i clinical trial at the national cancer center hospital, tokyo, japan the micelle carrier nk is based on peob polyaspartic acid block copolymers, in which the aspartic acid units were partially ca substituted with doxorubicin to form hydrophobic block the resulting substituted block copolymer forms micelles that are further noncovalently loaded with free doxorubicin preclinical studies in mice demonstrated higher nk activity against colon , m, and p, compared with the free drug moreover, nk has less side effects, resulting in less animal body and toxic death than the free drug clinically, the pluronic micelle formulation of doxorubicin has been most advanced based on the in vivo efficacy evaluation, pluronic l was selected for clinical development for the treatment of mdr cancers the final block copolymer formulation is a mixture of pluronic l and pluronic f, formulated in isotonic buffered saline this system contains mixed micelles of l and f, with an effective diameter of ca to nm and is stable in the serum prior to administration, doxorubicin is mixed nifedipine breast milk with this system, which results in spontaneous incorporation of the drug in the micelles the drug is easily released by diffusion after dilution of the micelles the formulation of doxorubicin with pluronic, spc, is safe, following systemic administration based on toxicity studies in animals a twosite phase i clinical trial of spc has been completed based on its results, the doselimiting toxicity of spc was myelosuppression, reached at mg m maximum tolerated dose was mgm phase ii study of this formulation to treat inoperable metastatic adenocarcinoma of the esophagus is near completion as well finally, phase i studies were reported for genexolpm, a cremophorfree polymer micelleformulated paclitaxel twentyone patient entered into this study with lung, colorectal, breast, ovary, and esophagus cancers no hypersensitivity reaction was observed in any patient neuropathy and myalgia were the most common toxicities there were partial responses the paclitaxel area under the curve and peak of the drug concentration in the blood were increased with the escalating dose, suggesting linear pharmacokinetics for genexolpm conclusions approximately two decades have passed since the conception of the polymer micelle conjugates and nanocontainers for drug delivery during the first decade, nifedipine breast milk only a few studies were published however, more recently, the number of publications in this field has increased tremendously during this period, novel biocompatible andor biodegradable block copolymer chemistries have been researched, the block ionomer complexes capable of incorporating dna and other charged molecules have been discovered, the ph and other chemical signal sensitive micelles have been developed many studies focused on the use of polymer micelles for delivery of poorly soluble and toxic chemotherapeutic agents to the tumors to treat cancer there has been considerable advancement in understanding the processes of polymer micelle delivery into the tumors, including passive and vectorized targeting of the polymer micelles notable achievements also include the studies demonstrating the possibilities for overcoming multidrug resistance in cancer, and enhancing drug delivery to the brain using block copolymer micelles systems overall, it is clear that this area has reached a mature stage, reinforced by the fact that several human clinical trials using polymer micelles for cancer drug delivery have been initiated at the same time, it is obvious that the possibilities for delivery of the diagnostic and therapeutic agents using polymer micelles are extremely broad, nifedipine breast milk and one should expect further increase in the laboratory and clinical research in this field during the next decade targeting polymer micelles to cancer sites within the body will address an urgent need to greatly improve the early diagnosis and treatment of cancer capabilities for the discovery and use of targeting molecules will support the development of multifunctional therapeutics that can carry and retain antineoplastic agents within tumors this will also be instrumental in developing novel biosensing and imaging modalities for the early detection of cancer and other devastating human diseases acknowledgment the authors acknowledge the support of the research using polymer micelles by grants from the national institutes of health ca, ns and eb, as well 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taillefer j, van lier je, lenaerts v and leroux jc optimizing ph responsive polymeric micelles for drug delivery in a cancer photodynamic therapy model drug targ yokoyama m, okano t, sakurai y, fukushima s, okamoto � and kataoka � selective delivery of adriamycin to a solid tumor using a polymeric micelle carrier system j drug targ greish k, nagamitsu a, fang j and maeda h copolystyrenemaleic acid pirarubicin micelles high tumortargeting efficiency with little toxicity bioconjug chem nasongkla n, shuai x, ai h, weinberg bd, pink j, boothman da and gao j crgdfunctionalized polymer micelles for targeted doxorubicin delivery angew chem int ed engl paulos cm, turk mj, breur gj and low ps folate receptormediated targeting of nifedipine breast milk therapeutic and imaging agents to activated macrophages in rheumatoid arthritis adv drug del rev yoo hs and park tg folate receptor targeted biodegradable polymeric doxorubicin micelles j control rel allen tm ligandtargeted therapeutics in anticancer therapy nat rev cancer gao z, lukyanov an, chakilam ar and torchilin vp pegpe phosphatidylcholine mixed immunomicelles specifically deliver encapsulated taxol to tumor cells of different origin and promote their efficient killing } drug targ tannock if and rotin d acid ph in tumors and its potential for therapeutic exploitation cancer res kataoka k, matsumoto t, yokoyama m, okano t, sakurai y, fukushima s, okamoto � and kwon gs doxorubicinloaded polyethylene glycolpolybetabenzyll aspartate copolymer micelles their pharmaceutical characteristics and biological significance } control rel yoo hs, lee ea and park tg doxorubicinconjugated biodegradable polymeric micelles having acidcleavable linkages } control rel hruby m, konak � and ulbrich � polymeric micellar phsensitive drug delivery system for doxorubicin, f control rel kabanov av, bronich tk, kabanov va, yu � and eisenberg a soluble stoichiometric complexes from polynethylvinylpyridinium cations and polyethylene 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venne a, alakhov v and kabanov a fundamental relationships between the composition of pluronic block copolymers and their hypersensitization effect in mdr cancer cells pharm res miller dw, batrakova ev and kabanov av inhibition of multidrug resistance associated protein mrp functional activity with pluronic block copolymers pharm res batrakova ev, li s, alakhov vy, elmquist wf, miller dw and kabanov av sensitization of cells nifedipine breast milk overexpressing multidrugresistant proteins by pluronic p pharm res minko t, batrakova e, li s, li y, pakunlu r, alakhov v and kabanov a pluronic block copolymers alter apoptotic signal transduction of doxorubicin in drugresistant cancer cells j control rel batrakova ev, han hy, miller dw and kabanov av effects of pluronic p unimers and micelles on drug permeability in polarized bbmec and caco cells pharm res evers r, kool m, smith aj, van deemter l, de haas m and borst p inhibitory effect of the reversal agents v, gf and pluronic l on mdr pgp, mrp and mrpmediated transport br j cancer batrakova ev, miller dw, li s, alakhov vy, kabanov av and elmquist wf pluronic p enhances the delivery of digoxin to the brain in vitro and in vivo studies ] pharmacol exp ther miller dw, batrakova ev, waltner to, alakhov v and kabanov av interactions of pluronic block copolymers with brain microvessel endothelial cells evidence of two potential pathways for drug absorption bioconjug chem batrakova ev, li s, elmquist wf, miller dw, alakhov vy and kabanov av mechanism of sensitization of mdr cancer cells by pluronic block nifedipine breast milk copolymers selective energy depletion br j cancer batrakova ev, li s, vinogradov sv, alakhov vy, miller dw and kabanov av mechanism of pluronic effect on pglycoprotein efflux system in bloodbrain barrier contributions of energy depletion and membrane fluidization } pharmacol exp ther batrakova ev, li s, alakhov vy, miller dw and kabanov av optimal structure requirements for pluronic block copolymers in modifying pglycoprotein drug efflux transporter activity in bovine brain microvessel endothelial cells f pharmacol exp ther rapoport n, marin ap and timoshin aa effect of a polymeric surfactant on electron transport in hl cells arch biochem biophys kabanov av, batrakova ev and alakhov vy an essential relationship between atp depletion and chemosensitizing activity of pluronic block copolymers ] control rel batrakova ev, li s, miller dw and kabanov av pluronic p increases permeability of a broad spectrum of drugs in polarized bbmec and caco cell monolayers pharm res batrakova ev, zhang y, li y, li s, vinogradov sv, persidsky y, alakhov v, miller dw and kabanov av effects of pluronic p on glut and mct transporters in the blood brain barrier pharm res in press kabanov av, batrakova nifedipine breast milk ev and miller dw pluronicr block copolymers as modulators of drug efflux transporter activity in the bloodbrain barrier adv drug del rev kabanov av and batrakova ev new technologies for drug delivery across the blood brain barrier curr pharm des kwon gs polymeric micelles for delivery of poorly watersoluble compounds crit rev ther drug carr syst adams ml and kwon gs relative aggregation state and hemolytic activity of amphotericin � encapsulated by polyethylene oxideblockpolynhexyl laspartamideacyl conjugate micelles effects of acyl chain length } control rel adams ml, andes dr and kwon gs amphotericin � encapsulated in micelles based on polyethylene oxideblockpolylamino acid derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity biomacromolecules croy sr and kwon gs the effects of pluronic block copolymers on the aggregation state of nystatin j control rel jagannath c, sepulveda e, actor jk, luxem f, emanuele mr and hunter rl effect of poloxamer crl on drug uptake and nitricoxidemediated killing of mycobacterium avium by macrophages immunopharmacology jagannath c, emanuele mr and hunter rl activity of poloxamer crl against drugsensitive and resistant strains of mycobacterium tuberculosis in macrophages and in mice nifedipine breast milk int j antimicrob agents jagannath c, emanuele mr and hunter rl activities of poloxamer crl against mycobacterium avium in macrophage culture and in mice antimicrob agents chemother jagannath c, wells a, mshvildadze m, olsen m, sepulveda e, emanuele m, hunter rl, jr and dasgupta a significantly improved oral uptake of amikacin in fvb mice in the presence of crl copolymer life sci torchilin vp pegbased micelles as carriers of contrast agents for different imaging modalities adv drug del rev trubetskoy vs, frankkamenetsky md, whiteman kr, wolf gl and torchilin vp stable polymeric micelles lymphangiographic contrast media for gamma scintigraphy and magnetic resonance imaging acad radiol trubetskoy vs, gazelle gs, wolf gl and torchilin vp blockcopolymer of polyethylene glycol and polylysine as a carrier of organic iodine design of long circulating particulate contrast medium for xray computed tomography } drug targ weissig v, whiteman kr and torchilin vp accumulation of proteinloaded long circulating micelles and liposomes in subcutaneous lewis lung carcinoma in mice pharm res katayose s and kataoka � watersoluble polyion complex associates of dna and polyethylene glycolpolyllysine block copolymer bioconj chem wolfert ma, schacht eh, toncheva v, ulbrich nifedipine breast milk k, nazarova � and seymour lw characterization of vectors for gene therapy formed by selfassembly of dna with synthetic block copolymers hum gene ther vinogradov sv, bronich tk and kabanov av selfassembly of polyamine polyethylene glycol copolymers with phosphorothioate oligonucleotides bioconjug chem itaka k, harada a, nakamura k, kawaguchi h and kataoka � evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions biomacromolecules roy s, zhang k, roth t, vinogradov s, kao rs and kabanov a reduction of fibronectin expression by intravitreal administration of antisense oligonucleotides nat biotechnol ogris m, steinlein p, kursa m, mechtler k, kircheis r and wagner e the size of dnatransferrinpei complexes is an important factor for gene expression in cultured cells gene ther oupicky d, ogris m, howard ka, dash pr, ulbrich � and seymour lw importance of lateral and steric stabilization of polyelectrolyte gene delivery vectors for extended systemic circulation mol ther haradashiba m, yamauchi k, harada a, takamisawa i, shimokado � and kataoka � polyion complex micelles as vectors in gene therapypharmacokinetics and in vivo gene transfer gene ther choi yh, liu f, parkjs nifedipine breast milk and kim sw lactosepolyethylene glycolgrafted poly llysine as hepatoma cell tapgeted gene carrier bioconjug chem vinogradov s, batrakova e, li s and kabanov a polyion complex micelles with proteinmodified corona for receptormediated delivery of oligonucleotides into cells bioconjug chem ward cm, pechar m, oupicky d, ulbrich � and seymour lw modification of plldna complexes with a multivalent hydrophilic polymer permits folatemediated targeting in vitro and prolonged plasma circulation in vivo } gene med nguyen hk, lemieux p, vinogradov sv, gebhart cl, guerin n, paradis g, bronich tk, alakhov vy and kabanov av evaluation of polyetherpolyethyleneimine graft copolymers as gene transfer agents gene ther gebhart cl, sriadibhatla s, vinogradov s, lemieux p, alakhov v and kabanov av design and formulation of polyplexes based on pluronicpolyethyleneimine conjugates for gene transfer bioconjug chem belenkov al, alakhov vy, kabanov av, vinogradov sv, panasci lc, monia bp and chow ty polyethyleneimine grafted with pluronic p enhances ku antisense delivery and the ionizing radiation treatment efficacy in vivo gene ther yokoyama m, miyauchi m, yamada n, okano t, sakurai y, kataoka � and inoue s characterization and anticancer activity of the micelleforming polymeric anticancer drug nifedipine breast milk adriamycinconjugated polyethylene glycolpolyaspartic acid block copolymer cancer res nakanishi t, fukushima s, okamoto k, suzuki m, matsumura y, yokoyama m, okano t, sakurai y and kataoka � development of the polymer micelle carrier system for doxorubicin } control rel danson s, ferry d, alakhov v, margison j, kerr d, jowle d, brampton m, halbert g and ranson m phase i dose escalation and pharmacokinetic study of pluronic polymerbound doxorubicin spc in patients with advanced cancer br } cancer valle jw, lawrance j, brewer j, clayton a, corrie p, alakhov v and ranson m a phase ii, window study of spc as firstline therapy in inoperable metastatic adenocarcinoma of the oesophagus asco annual meeting vol abstract no kim ty, kim dw, chung jy, shin sg, kim sc, heo ds, kim nk and bang yj phase i and pharmacokinetic study of genexolpm, a cremophorfree, polymeric micelleformulated paclitaxel, in patients with advanced malignancies clin cancer res this page is intentionally left blank vesicles prepared from synthetic amphiphiles � polymeric vesicles and niosomes ijeoma florence uchegbu and andreas g schatzlein introduction this chapter will examine what is known about vesicles prepared from synthetic nifedipine breast milk amphiphiles and will encompass a review of the data published on polymeric vesicles and nonionic surfactant vesicles niosomes schematic representations of the molecular arrangements in these systems are as depicted in fig examples of drug delivery applications will also be presented vesicular systems arise when amphiphilic molecules self assemble in aqueous media in an effort to reduce the high energy interaction between the hydrophobic portion of the amphiphile and the aqueous disperse phase, and maximize the low energy interaction between the hydrophilic head group and the disperse phase fig these self assemblies reside in the nanometre to micrometre size domain excellent reviews exist on the self assembly of amphiphiles and hence this topic will not be dealt with in great detail here vesicles are important pharmaceutical systems, especially as liposomes, the result of phospholipid self assembly, are licensed for the clinical delivery of anti cancer drugs it is thus possible that the vesicles described here may be incorporated into licensed medicines at some point in future � � s � � � luvj � � � b self assembling amphiphilic polymers self assembling polymerisable monomers polymerisation a polymerised vesicles nifedipine breast milk � �� �� u � d self assembling non ionic surfactant vesicles niosomes q � polymer backbone hydrophilic head group ���� hydrophobic tail c polymeric vesicles prepared from self assembling cetyl polyethylenimine amphiphiles, arrow shows polymer coat fig polymeric vesicles and niosomes polymeric vesicles arise from a the self assembly of polymerizable monomers which are subsequently polymerized and b and c the self assembly of amphiphilic polymers bearing hydrophobic pendant groups additionally, both block and random copolymers are able to self assemble into polymeric, vesicles, d niosomes arise from the self assembly of nonionic surfactants polymeric vesicles polymeric vesicles were first investigated as a means of stabilizing the metastable self assemblies formed from low molecular weight amphiphiles, with the polymer providing a kinetic trap for the self assembled system a wealth of polymer architectures are now known to assemble into vesicles namely block copolymers, random graft copolymers, polymerized self assembling monomers and polymers bearing lipid pendant groups, fig additionally, polymeric vesicles, although not normally termed as such, arise from the self assembly of amphiphilic polymers, ie polyoxyethylene amphiphiles with a lipids to give polyoxyethylene coated liposomes, or b nonionic surfactants nifedipine breast milk to give polyoxyethylene coated niosomes a summary of the liposome literature exists in other parts of this volume and hence liposomepolymer interactions or block copolymers random copolymers water soluble polymer with hydrophobic pendant groups in � lifilil fitfffiffll hydrophobic monomers polymer backbone attached to hydrophobic pendant groups via a hydrophilic spacer hydrophobic pendant groups hydrophilic spacer hydrophilic monomersf hydrophobic pendant group fig schematic representation of self assembling vesicle forming polymers liposomes incorporating minor amounts of polymers will not be treated in this chapter while details on niosomes appear later on this chapter the current section of this chapter will thus focus on the self assembly of block copolymers, random graft copolymers, polymers bearing lipid pendant groups and polymerized vesicles in which polymerization takes place after self assembly polymer self assembly as may be inferred from the list above, there are two main ways in which polymeric vesicles may be formed, either from the self assembly of polymers or from the polymerization of monomers subsequent to self assembly fig vesicular self assembly is not a spontaneous process and normally requires an input of energy in the form of probe sonication nifedipine breast milk for instance it is known that vesicle formation from low molecular weight amphiphiles in aqueous media is controlled by two opposing forces, namely the steric or ionic repulsion between hydrophilic head groups which maximizes the interfacial area per molecule and the attractive forces between hydrophobic groups which serve to reduce the interfacial area per molecule attaining a minimal interfacial energy is thus served by the formation of a closed spherical bilayer in general terms, the lesser hydrophobic amphiphiles form micelles, while amphiphiles of intermediate hydrophobicity form vesicles the self assembly of polymers into vesicles is governed by similar constraints and as such, the hydrophobiclipophilic balance of polymers determines whether a polymer will self assemble into vesicles in a similar manner as is found in liposomes and niosomes there are also some polymer specific factors which impact on vesicle forming ability for example, the degree of polymerization is critical to vesicle forming ability, and generally very high degrees of polymerization prevent vesicle formation furthermore, the flexibility of the hydrophobic block in block copolymers determines which self assemblies will be formed the more flexible hydrophobic portions of the polymer are able nifedipine breast milk to form vesicles, whereas the more rigid polymers are unable to self assemble into three dimensional structures polymeric vesicles appear to be largely unilamellar, and unilamellarity is favored when the molecular weight of the amphiphile increases polymeric vesicles often possess superior mechanical stability, are usually less susceptible to degradation by organic solvents and soluble surfactants, and are frequently less permeable to hydrophilic solutes, compared with vesicles prepared from low molecular weight amphiphiles, the widespread exploitation of these fascinating nanocarriers for the development of responsive and biomimetic nanomedicines with superior stability characteristics is what awaits the science polymers bearing hydrophobic pendan t groups the first report on the use of preformed polymers to prepare polymeric bilayer vesicles was presented in by kunitake and others in this report, bilayer vesicles were prepared from compound shown in fig a compound comprises a hydrophilic polyacrylamide backbone and dialkyl hydrophobic pendant groups separated from the polymer backbone by hydrophilic oligooxyethylene spacers, as shown schematically in fig the hydrophilic spacer group between the dialkyl moieties is essential for vesicle formation for these polyacrylamide type polymers the hydrophilic spacer allows the decoupling of the polymer motion nifedipine breast milk from the ordering of the bilayer the introduction of essentially water soluble carbohydrate [eg compound , fig a], polyelectrolyte [eg compounds , fig a] and polyamino acid [eg compound , fig a] polymer backbones bearing hydrophobic pendant groups is a fairly recent development the bilayer arrangement is as depicted in fig b and the thick polymer coat is clearly visible on micrographs fig for drug delivery applications, it is important to appreciate that the conversion of the polyllysine into vesicles reduces its cytotoxicity, thus allowing this molecule to be exploited as a pharmaceutical excipient oh fig a representative vesicle forming polymers from the polyacrylamide compound , chitosan compound poly amine compounds la and poly a mine acid compound n classes of polymers bearing hydrophobic pendant groups ��� vesicle bilayer arrangement lipid pendant groups cholesterol former backbone fig b the glycol chitosan polymer ie compound , is arranged in the bilayer as shown polyethylenimine [compound , fig a] and polyllysine [compound , fig a] based amphiphiles have been studied in some detail and the formation of vesicles from these amphiphiles is dependent on the level of lipid pendant groups hydrophobically modified polyethylenimine for example forms dense nanoparticles, nifedipine breast milk bilayer vesicles or micellar self assemblies, depending on its hydrophobic content hence, polyethylenimine amphiphiles with a hydrophobic content of and above favor dense nanoparticle self assemblies, while a hydrophobic content of favors bilayer vesicle assemblies, and finally a hydrophobic content of less than favors the formation of micellar assemblies a remarkably similar trend has been reported for the polyoxyethylenewoca polylactic acid system in that a polylactic acid fraction of favors dense nanoparticles, while a polylactic acid fraction of favors bilayer vesicle assemblies, and a poly lactic acid fraction of less than favors the production of micellar self assemblies the sizes of the vesicle and dense nanoparticle assemblies formed from amphiphilic polyethylenimines are also dependent on polymer levels of hydrophobic modification mole cetylation and the relationships shown in eqs and have been developed dv = ct dn = ct where dv = vesicle zaverage mean hydrodynamic diameter, ct = mole cetylation number of cetyl groups per monomer units, and dn = nanoparticle zaverage mean hydrodynamic diameter the molecular weight of the polymer is also an important factor to consider when choosing vesicle forming polymers the importance of this parameter has nifedipine breast milk been demonstrated with the polyllysine vesicle system [eg compound , fig a] with these amphiphiles a vesicle formation index f has been computed where h = mole unreacted llysine units, l = mole llysine units substituted with palmitic acid and dp = the degree of polymerisation of the polymer an f value in excess of is necessary for vesicle formation additionally, not only does the molecular weight of the polymer impact on vesicle formation, but it is also a direct controller of the vesicle mean size the relationship shown in eq has been developed for the palmitoyl glycol chitosan system vm = dv where mw = polymer molecular weight, and dv = vesicle zaverage mean hydrodynamic diameter block copolymers fig examples of some vesicle forming block copolymers compound , compound , and compound g block copolymer vesicles, termed polymersomes are fairly new discoveries, being first reported in the s polymersomes have been prepared from a variety of block copolymers, some examples of which are given in fig there is a clear relationship between the hydrophobic content of polymers and self assembly low levels of hydrophobicity less than of the polymer consisting of nifedipine breast milk hydrophobic moieties favors the formation of micelles and intermediate levels of hydropho bicity favors the formation of bilayer vesicles, for the self assembly of block copolymers, it has been established that generally the critical packing parameter cpp cpp al should approach unity for vesicular self assemblies to prevail, where v = volume of the hydrophobic block, = length of the hydrophobic block and a = the area of the hydrophilic block vesicle sizes are varied and range from tens of nanometres to tens of microns polymersome membranes are nm thick times thicker than the nm membrane thickness displayed by conventional low molecular weight amphiphiles the thickness of the membrane is determined by the degree of polymerization in the hydrophobic block and these extra thick membranes confer, on the vesicle, exceptional stability to soluble surfactants and mechanical stress, with these vesicles, there is an asymmetric distribution of the polymers in the inner and outer leaflets of the bilayer and polymers with a large hydrophilic chain length are preferentially localized to the exterior leaflet and vice versa preferred residence in the outer leaflet is favored by the more hydrophilic polymers, because nifedipine breast milk the greater repulsion between the longer hydrophilic corona molecules on the outer leaflet stabilize the vesicle curvature vesicle stability is a desirable characteristic for pharmaceutical vesicles and as such, a great deal of effort has been expended on producing stable systems as the drive for nanomedicines medicines incorporating functional nanoparticles grows, stability issues will need to be adequately addressed to ensure the widespread adoption of such systems in actual fact, the early workers in the polymeric vesicle field were primarily driven by this need to produce stable drug carriers extremely stable systems are possible on polymerization of block copolymers subsequent to self assembly polyethylene oxidemoccpolyftrimethoxysilylpropyl methacry late] copolymer vesicles in water, methanol, triethylamine mixtures produced polymerized polymersomes that are stable for up to one year triethylamine hydrolyzes the trimethoxysilyl groups and then catalyzes their polycondensation to yield an extremely stable hydrophobic polysilsesquioxane core, additionally, polyethylene oxidewocfcpolybutadiene vesicles on cross linking produce vesicles which are organic solvent resistant preparing vesicles from selfassembling polymers polymeric vesicles are relatively simple to prepare the input of energy is achieved in the laboratory by probe sonication of the amphiphilic polymer in the disperse phase nifedipine breast milk however, clearly the energy required for self assembly is not trivial as vesicles are not easily formed by hand shaking, unlike low molecular weight surfactant formulations vesicles once formed are morphologically stable for months and may be loaded with hydrophilic and hydrophobic [see fig b below] solutes, by probe sonicating in the presence of such solutes commercially, it is envisaged that polymeric vesicles may be fabricated by microfluidization and high pressure homogenization techniques self assembling polymerizable monomers polymerized vesicles may also be prepared by utilizing self assembling polymerizable amphiphiles, followed by the polymerization of the resulting vesicular self assembly fig examples of some polymerizable vesicle forming monomers are shown in fig this method of producing polymerised vesicles is the oldest form of polymeric vesicle technology fig polymerizable vesicle forming monomers used to make polymerized vesicles by jung and others compound , cho and others compound ll, hub and others compound and bader and others compound polymerized vesicles prepared using polymerized self assembling monomers are essentially polymer shells and it is unclear how much of the bilayer assembly actually survives the polymerization step the advantage, however, is that they are extremely nifedipine breast milk stable, resisting degradation by detergents or organic solvents they are also less leaky, thermostable, and because the vesicle forming components are kinetically trapped by the polymerization process, they have improved colloidal stability a major advantage of these nanosystems is that they may be isolated as dry powders which are readily dispersible in water to give nm particles thus potentially allowing the formulation of solid vesicle dosage forms polymerization involves fairly reactive species and hence vesicles are best prepared prior to drug loading, which may be a limitation polymeric vesicle drug delivery applications polymeric vesicles, which are the focus of this chapter, exist in two main varieties as illustrated in fig these technologies are suitable candidates for the development of robust, controllable and responsive nanomedicine drug carriers drug targeting polyoxyethylene amphiphiles, when incorporated into liposomal and niosomal bilayers, prolong vesicle circulation and facilitate tumor targeting, due to the leaky nature of the poorly developed tumor vascular endothelium only mole polyethylene oxide � lipid amphiphiles may be incorporated into liposomes or niosomes without a loss of vesicle integrity due to the preferred tendency of the hydrophilic polyoxyethylene amphiphiles to form micelles poly nifedipine breast milk mersomes composed of polyethylene oxidewocfcpolybutadiene or polyethylene oxidewocfcpolyethylethylene, in which the entire vesicle surface is covered with the polyethylene oxide coat, have been studied as long circulating nanocarriers for drug delivery the circulation time of polyethylene oxide polymersomes is directly dependent on the length of the polyethylene oxide block and polymer some half lives of up to hrs have been recorded in rats with a polyethylene oxide degree of polymerization of this half life compares favorably with a half life of hrs recorded for polyoxyethylene coated liposomes it is assumed that the surface coverage of the polymeric vesicles is responsible for the reduced clearance of these polymersomes from the blood the long half life of these polymersomes makes them excellent candidates for the development of anti tumor medicines furthermore, drug release may be controlled in the polymersomes by controlling the hydrolysis rate of the hydrophobic blocks this has been demonstrated with polyllactic acidbocfcpolyethylene glycol and polycaprolactonemocc polyethylene glycol vesicles hydrolysis of the hydrophobic block causes the polymer to move from a vesicular to a micellar assembly, as the overall level of hydrophobic content diminishes, and this in turn leads nifedipine breast milk to drug release hydrolysis rates and implicitly release rates may be controlled by varying the relative level of the hydrophobic blocks carbohydrate polymeric vesicles may also be used as drug targeting agents vesicles prepared from glycol chitosan vesicles improve the intracellular delivery of hydrophilic macromolecules and anti cancer drugs, the latter is achieved with the help of a transferrin ligand attached to the surface of the vesicle gene delivery polyllysine based vesicles, prepared from compound [fig a] have been used for gene delivery as these vesicles are less toxic than unmodified polyllysine and produce higher levels of gene transfer table l the production of polymeric vesicles and the resultant reduction in cytotoxicity enables polyllysine to be used in in vivo gene, as the unmodified polymer is too toxic for in vivo use when the targeting ligand, galactose, was bound to the distal ends of the polyoxyethylene chains, gene expression was increased in hepg cells in vitro however, in vivo targeting to the liver hepatocytes was not achieved with these systems a similar procedure with the polyethylenimine vesicles prepared using compound [fig a] also resulted in a reduction in the nifedipine breast milk cytotoxicity of the polymer table l, although in this case, the polyethylenimine vesicles were not as efficient gene transfer agents as the free polymer table biological activity of polyethylenimine and polyllysine vesicles polymer a cells a ic gene transfer ic gene transfer xg ml relative to parent jxgml relative to parent polymer polymer polyethylenimine polymer fig a polymer , cholesterol vesicles gg polyllysine polymer fig a responsive release the ultimate goal of all drug delivery efforts is the simple fabrication of responsive systems that are capable of delivering precise quantities of their pay load in response to physiological or more commonly pathological stimuli preprogrammable pills, implants and injectables are so far merely the unobtainable ideal, however, polymeric systems have been fabricated with responsive capability and it is possible that in the future, these may be fine tuned to produce truly intelligent and dynamic drug delivery devices or systems the various environmental stimuli that may be used to trigger the release of encapsulated drug are outlined below and examples are given of existing developments in the area however, in addition to the areas covered below, it may be possible in future nifedipine breast milk for pathology specific molecules to interact with polymeric vesicles to trigger release ph diblock polypeptides, in which the hydrophilic block consists of ethylene glycol derivatised amino acids llysine, and the hydrophobic block consists of poly lleucine, form ph responsive vesicles which disaggregate at low ph, providing the level of lleucine and polymer chain length is maintained within defined limits of about mole and the polymer has a degree of polymerization of less than these llysine based systems may be applied to facilitate endosome specific release enzymatic vesicles which release their contents in the presence of an enzyme may be formed by loading polymeric vesicles with an enzyme activated prodrug fig the particulate nature of the drug delivery system should allow the drug to accumulate in tumors, for example, where it may then be activated by an externally applied enzyme in a similar manner to the antibody directed enzyme prodrug therapeutic strategy the antibody directed enzyme prodrug therapeutic strategy enables an enzyme to be homed to tumors using antibodies followed by the application of an enzyme activated prodrug alternatively, a membrane bound enzyme may be used to control and ultimately nifedipine breast milk prolong the activity of either an entrapped hydrophilic drug entrapped in the vesicle aqueous core or an entrapped hydrophobic drug entrapped in the vesicle membrane as illustrated in fig it is possible that the enzyme may be chosen such that it is activated in the presence of pathology specific molecules, thus achieving pathology responsive and localized drug activity t � ?� vesicle bound enzyme external substrate � external enzyme vesicle loaded substrate a� control solution substrate � �i� �i� enzymetjme min a water soluble substrate fig a enzyme activated polymeric vesicles vesicles bearing membrane bound enzyme i were formed by probe sonicating compound [fig a], cholesterol, nbiotinylated dipalmitoyl phosphatidyl ethanolamine gg in neutral phosphate buffer ml, isolation of the vesicles by ultracentrifugation , g, redispersion in a similar volume of neutral phosphate buffer and incubation of the vesicles with sgalactosidase strepta vidin u membrane bound enzyme ml was then incubated with onitrophenyljd galactoside mm, ml and the absorbance monitored � = nm the control solution contained similar levels of substrate onitrophenyljsdgalactoside but no enzyme vesicles encapsulating nitrophenylidgalactoside ii were prepared by probe sonicating compound , cholesterol gg in the presence of nifedipine breast milk onitrophenyljdgalactoside solution mm, ml and isolation of the vesicles by ultracentrifugation and redispersion in neutral phosphate buffer these latter vesicles ml were then incubated with dgalactosidase uml, ml and the absorbance once again monitored magnetic magnetically responsive polymerized liposomes composed of ,di , octadecadienoylsnglycerolphosphorylcholine, loaded with ferric oxide and subsequently polymerized may be localized by an external magnetic field to the small intestine, and specifically the payers patches these polymerized vesicles are stable to the degradative influence of solubilizing surfactants such as tritonx , and hence should not suffer excessive bile salt mediated degradation during gut transit these magnetically responsive polymeric vesicles improve the absorption of drugs via the oral route membrane bound enzyme hydrophobic substrate fig b enzyme activated polymeric vesicles vesicles bearing membrane bound enzyme and containing the hydrophobic substrate fluorescein disdgalactospyranoside were formed by probe sonicating compound [fig a], cholesterol, nbiotinylated dipalmitoyl phosphatidyl ethanolamine, fluorescein didgalactospyranoside g g in neutral phosphate buffer ml and incubation of the resulting vesicles with bgalactosidase streptavidin u the fluorescence of the enzyme hydrolysed substrate was then monitored excitation wavelength = nm, emission wavelength = nm oxygen block copolymer vesicles which are destabilized nifedipine breast milk by oxidative mechanisms have been constructed from polyoxyethylenemoccpolypropylene sulphidewocc polyoxyethylene aba block copolymers these polymeric vesicles are destabilized on the oxidation of the central sulphide block to give sulphoxides and ultimately sulphones on oxidation, vesicles are transformed to wormlike micelles and finally to spherical micelles, eventually releasing their contents nonionic surfactant vesicles niosomes self assembly min int the self assembly of nonionic surfactants into niosomes is dependent on the hydrophilic � hydrophobic balance of the surfactant and a cpp eq of between enables niosomal self assembly some examples of niosome forming molecules are given in fig further molecular specifics that govern niosome fig b niosomal membrane additives, compound = cholesterol, compound = solulan c formation by nonionic surfactants may be found in published reviews, compounds such as compounds from the sorbitan surfactant class are established pharmaceutical excipients, and hence formulation scientists looking to prepare a niosome formulation for speedy transition to the clinic will do well looking at this class of molecules for exploitable materials most niosomes will not only contain the nonionic surfactant, but will also contain other molecules such as the membrane stabilizer cholesterol [fig b] the nifedipine breast milk bilayer membrane is an ordered structure which may exist in the gel or liquid crystal state essentially, molecules are more mobile in the liquid crystalline state, enjoying lateral diffusion within the bilayer that is denied them in the gel state for any system, the liquid crystal state exists at a higher temperature t than the gel state an increase in temperature favors the transition from the gel to the liquid state because of the entropy gain as associated with this transition, ultimately leading to a lowering of the free energy ag of the system cholesterol abolishes this membrane phase transition, thus fluidizing the gel state niosomes are nm to �� in size and often their surfaces must be stabilized against aggregation molecules such as the cholesteryl polyoxyethylene ether � solulan c compound , fig b or the ionic molecule dicetyl phosphate have been used to confer steric and electrostatic stabilization on these vesicles respectively the reader should be aware that the inclusion of minor quantities by actual weight or molar content of ionic surfactant does not prevent these structures from being discussed in this chapter under the niosome heading niosomes nifedipine breast milk are often formulated with minor quantities of cationic and other surfactants it can be said that the formulation of liposomes with polyethylene oxide amphiphiles such as distearolyphosphatidylethanolaminepolyethylene glycol was the crucial step that allowed liposomes to become clinically relevant drug delivery systems the resulting liposomes possess a hydrophilic polymer surface, which prevents recognition and clearance of the particles from the blood by the liver and spleen macrophages thus increasing the liposomes circulation time and allowing tumor targeting niosomes nonionic surfactant vesicles, when formulated with a water soluble polyoxyethylene cholesteryl ether � solulan c, also circulate for prolonged periods in the blood, accumulate in the tumor tissue and improve tumoricidal activity as well as stabilizing vesicles in the blood, polyoxyethylene amphiphiles also stabilize vesicles against aggregation, thus promoting vesicle colloidal stability polyoxyethylene amphiphiles, such as solulan c, have a large hydrophilic head group [fig b], and are thus more hydrophilic than the vesicle forming amphiphiles, and hence the level of the former must be kept low to avoid solubilization of the membrane and the formation of mixed micelles in actual fact unusual morphologies result from the incorporation of nonmicellizing quantities nifedipine breast milk of solulan c in vesicles as discussed below polyhedral vesicles and giant vesicles discomes a series of unusual morphologies have been isolated from the hexadecyl diglyc erol ether, solulan c, cholesterol phase diagram [fig a] the addition of solulan c to hexadecyl diglycerol ether [compound , fig a] niosomes eventually results in the formation of mixed micelles at submi cellar concentrations of solulan c mole, however, giant vesicles discomes of m in size are formed discomes arc therm ores ponsive vesicles, which become more leaky as the temperature is increased from room temperature to �c these vesicles may thus be used to construct ther mores ponsive controlled release systems in cholesterol low regions of the hexadecyl diglycerol ether, cholesterol, solulan c phase diagram, polyhedral vesicles [figs a and b] are found these polyhedral vesicles are able to entrap water soluble solutes and the membrane, which is in the gel state contains areas of high and low curvature as shown in polyhedral vesicles pm spherical, helical, tubular vesicles ftm discomes jim small spherical & helical vesicles fim discomes firri mixed micelles � � g @ � � � � cholesterol crystals spherical vesicles ?