free rd �� free rd blank cscoated nanocapsules h rdloaded cscoated nanocapsules time h fig rhodamine rd amount transported across the rabbit cornea in an ex vivo study for rdloaded chitosan cs coated nanocapsules and control formulations consisting of a rd solution and a physical mixture of free rd and blank cscoated nanocapsules statistically significant different from free rd statistically significant different from free rd and free rd plus blank cscoated nanocapsules reprinted from ref , with permission from elsevier ai cd � � � � ?� � � � � ?� � ?� � q � � � � extent than the noncoated pecl nanocapsules, a result that could be attributed to the increased surface retention of the nanocapsules in the mucus prevacid free powered by vbulletin layer therefore, overall, the results obtained until now with nanoparticulate carriers coated with mucoadhesive polymers permit us to conclude the efficacy of this approach, in terms of increasing the retention of the nanoparticles at the eye surface this improved retention could be translated depending on the solubility properties of the drug encapsulated to a more important corneal penetration, or in a greater retention on the ocular surface polyethyleneglycol pec coating a very different alternative in the coating approach has been the one intended to provide the nanoparticulate carrier with an improved stability upon contact with the mucosal fluids in fact, both the mucus layer and the lachrymal fluids are very rich in enzymes and proteins, which may be attached to the nanoparticles and prevacid free powered by vbulletin promote their aggregation polyethylene glycol peg appears to be an ideal candidate for such purpose, since it has been widely used to prevent the interaction of colloidal carriers with proteins for example, in a study performed by us, we observed that a peg coating around pla nanoparticles prevented their aggregation in the presence of lysozyme highly concentrated in the mucus layer on the other hand, this stabilizing effect has been the main explanation for the successful behavior of pegcoated pla nanoparticles as transmucosal drug carriers ie after nasal and oral administration, therefore, from these studies we suggested that the presence of a hydrophilic peg layer onto the surface of polyester nanoparticles could result in an enhanced stability and, hence, to an improved interaction of prevacid free powered by vbulletin these nanosystems with the ocular mucosa the first report on the positive effect of the peg coating approach on ocular drug administration was published by fresta et al these authors evaluated the ocular bioavailability of acyclovirloaded pegcoated ���� nanoparticles and observed a significant increase of the drug levels in the aqueous humor, when comparing these systems with an aqueous acyclovir suspension and with a physical mixture of the free drug and the blank pegcoated ���� nanoparticles interestingly, in this work, the authors did not consider the possibility of an improved stability rather, they suggested that the pegcoated particles might have an improved mucoadhesion however, no studies were reported to verify this mechanistic hypothesis in a more recent work, the same group reported the prevacid free powered by vbulletin efficacy of the peg coating but with a different core pla nanoparticles, in terms of increasing the ocular bioavailability aqueous humor drug levels of acyclovir moreover, these authors observed that the positive effect of the peg coating disappeared, when the mucus layer was removed using nacetylcysteine, prior to the administration of these systems to rabbits this observation led the authors to suggest that the mucoadhesion of the pegcoated nanocarriers may play a role in its mechanism of action however, this result could also be understood as a consequence of the stabilizing effect provided by peg in the mucosal surfaces this effect being not visible in the absence of mucus in order to obtain further insights into the interaction of pegcoated nanoparticles with the prevacid free powered by vbulletin cornea, we have recently compared their behavior with that of the uncoated pecl nanocapsules and the chitosancoated nanocapsules the confocal images showed that the three types of nanocapsules were able to enter the corneal epithelium however, their penetration depth followed the ranking of peg coated nanocapsules uncoated nanocapsules chitosancoated nanocapsules the more important corneal penetration of pegcoated nanocapsules, as compared with that of the noncoated ones, was suggested to be a consequence of their improved stability in the mucosal fluids the chitosan coating is also known to affect the stability of colloidal particles positively in the presence of the proteins such as lysozyme however, in this case, the superficial retention of chitosan coated nanocapsules described above could also be understood as a result of prevacid free powered by vbulletin their mucoadhesive character overall, the results obtained so far with nanoparticulate drug carriers, coated with hydrophilic polymers, indicate that depending on their nature, these polymers are able to increase the stability andor the mucoadhesion of the nanocarrier it could also be presumed that an increase in the mucoadhesion should lead to an accumulation of the drug carrier at the ocular surface also, in the case of drugs with adequate permeability properties, it should lead to an increase and prolongation in the corneal penetration similarly, an increase in the stability is expected to lead to a more important interaction and transport of the nanoparticles across the corneal epithelium moreover, these results suggest that both the extent of the interaction and the penetration depth of prevacid free powered by vbulletin the nanocarriers with the cornea, can be modulated by providing them with an appropriate coating despite the need of additional mechanistic studies as evidence, the results reported so far provided some basis for the development of strategies intended as an efficient drug targeting to specific ocular structures, as discussed below third nanoparticles generation towards functionalized nanocarriers the new tendency in the design of the new drug delivery systems is directed towards integrating several drug delivery technologies, in order to provide the system with unique properties in the particular case of the ocular drug delivery, the design of highly sophisticated drug delivery nanosystems could benefit from the knowledge gained from the application of such systems to other trasmucosal routes of administration as described in the prevacid free powered by vbulletin previous chapters of this book, some of the present efforts on the design of more specialized nanocarriers go through func tionalizing their surface this means that the design of nanoparticles with surface characteristics allowing their functionalization with specific targeting moieties, is able to selectively direct the nanocarrier to the predetermined ocular structures among the targeting moieties described till now, lectins may represent an interesting option for targeting the ocular mucosa lectins are glycoproteins capable of recognizing and binding reversibly to specific carbohydrate moieties which are present on cell surfaces and mucin in fact, lectinlike molecules are known to be important in the adhesion of microorganisms to mucosal surfaces therefore, lectins clearly differ from conventional mucoadhesive materials which interact non specifically with the mucus prevacid free powered by vbulletin or simply adhere to biological surfaces some examples of lectins are wheat germ agglutinin and phasoleus vulgaris agglutinin, which bind specifically to nacetylgalactosamine and mannose receptors, respectively with regard to the potential of lectins as targeting moieties for ocular drug delivery, it is a known fact that lectins can bind to the corneal and conjunctival surfaces and also to some constituents of the tear film from our knowledge, despite this information, there is no evidence of the potential of the targeted systems in the ocular drug delivery field a different category of targeting ligands is represented by the monoclonal antibodies the initial attempts towards the monoclonal antibodybased targeting approach have been directed to the treatment of ocular herpes simplex virus hsv infection more prevacid free powered by vbulletin specifically, norley et alm proposed the attachment of monoclonal antibodies antiglycoprotein d of hsv to liposomes, in order to achieve the targeted delivery of antiviral drugs to the infected corneal cells the results of this work showed the ability of these immunoliposomes to preferentially bind to virusinfected corneal cells in vitro however, there are no in vivo data available to support the efficacy of this targeting strategy, thus far the lack of reported success of the monoclonal antibodybased targeting approach could be related to the late clinical development of antibodies nevertheless, the enormous effort devoted to the development of antibodies for therapeutic or diagnosis purposes in the last few years opens optimistic prospects with regard to their use as targeting moieties for nanoparticulate carriers prevacid free powered by vbulletin within this context, pegcoated nanoparticles offer interesting opportunities for the functionalization with ligands such as lectins and monoclonal antibodies additionally, as polysaccharides present many available reactive groups, active targeting could also be attained by grafting ligands onto the polysaccharidecoated nanoparticles nanoparticulate polymer compositions as subconjuctival drug delivery systems the subconjunctival route has been proposed as an alternative to the topical drug delivery route, in order to force the retention of a significant amount of drug in the eye the drug molecules locate underneath the conjunctival epithelium are supposed to diffuse through the sclera and reach the inner eye there is no doubt that the most important limitation of this modality of administration is the poor acceptability by the patients therefore, the use of controlled release micro and nanoparticles was thought to be a good approach in order to reduce the number of injections despite the logic of this approach, no improved pharmacological andor therapeutical effects have been reported so far for either micro or nanoparticles for example, the subconjunctival injection of cyclosporinloaded plga microparticles failed to improve the response of this drug on the other hand, to the best of our knowledge, there has been no report on the efficacy of nanoparticles for the delivery of drugs at the subconjunctival level the study reported by amrite et al showed that the model fluorescent nanoparticles nm and microparticles �m administered subconjunctival were not able to cross the sclera, remaining at the injection site nanoparticulate polymer compositions as prevacid free powered by vbulletin intravitreal drug delivery systems most diseases affecting the posterior segment of the eye are chronic in nature and require prolonged drug administration these diseases are one of the major causes of blindness in the developed world unfortunately, the described difficulty of reaching effective drug levels at intraocular structures represents a major limitation associated to these therapies ie treatment of proliferative vitreoretinopathy, endophthalmitis, recurrent uveitis, acute retinal necrosis, choroidal neovascularization and cytomegalovirus retinitis in these severe situations, the intravitreal injection becomes the route of choice for drug delivery however, in clinical practice, this modality of administration has important drawbacks i poor patient acceptability, which may lead to failure of therapy ii rapid drug elimination from vitreous humor ie removal to the systemic circulation along with prevacid free powered by vbulletin the aqueous humor drainage, active secretion from the retina iii possible retinal toxicity of certain potent drugs iv potential hazards associated with repeated intravitreal injection, such as the clouding of the vitreous humor, retinal detachment, lens damage and endophthalmitis the above indicated problems illustrate the need for the design of adequate controlled release systems which could minimize the frequency of injection vitrasert� is an example of a commercially available sustained release intraocular device for ganciclovir, which has been approved for use in patients suffering from cytomegalovirus cmv this implant is a reservoir system consisting of a magnesium stearate core containing the drug, and a coating of ethylenevinyl acetate polymers apart from the necessity of surgical removal, additional problems observed for this device include prevacid free powered by vbulletin endophthalmitis, retinal detachment, dislocation of implant and poor intravitreal drug levels due to its placement in the suprachoroidal space within this context, biodegradable micro and nanoparticles appear to offer advantages when compared with large devices, since they can be injected through a needle, thus avoiding the necessity of a surgical procedure among the particulate carriers investigated to date for intraocular drug delivery, those made of biodegradable polyesters such as polylacticglycolic acid plga are expected to offer a significant potential in fact, there are already a number of reports on the biodegradability, tolerability and efficacy of plga intraocular implants, and microparticles for a review, see ref with regard to the specific potential of nanoparticles, in two studies published in aimed at evaluating the interaction of prevacid free powered by vbulletin pla microparticles im with retinal pigment epithelium cells, it was found that these particles were internalized by the above mentioned cells this finding was justified by the known phagocytic activity of these cells, which, on the other hand, are essential for the maintenance of retinal metabolism and visual acuity this initial observation was corroborated in a more recent study which evidenced that plga nanoparticles were localized within these cells even at months postadministration moreover, these authors observed that plga nanoparticles were well tolerated following intravitreal injection to rats therefore, these publications showed the potential of nanoparticles no only as simple depot controlled release systems but as intracellular controlled delivery systems for bioactive molecules surprisingly, despite the attractive features of plga nanoparticles as intraocular prevacid free powered by vbulletin delivery systems, the information reporting the success of this approach is scarce for example, in a very recent publication it was shown that plga nanoparticles could work as gene delivery systems to the posterior segment of the eye concretely, the plasmid encoding the red nuclear fluorescent protein rnfp was associated to plga nanoparticles and then injected into the vitreous cavity of rabbits the results showed an important level of rnfp expression within the retinal pigment epithelial cells, thus indicating the adequate internalization and delivery of the plasmid into the cells on the basis of these findings, these authors suggested the potential of nanoparticles for designing future genebased ocular therapy strategies another type of nanoparticles that has been investigated for intravitreal drug delivery is prevacid free powered by vbulletin the one consisting of ���� more specifically, these nanoparticles were tested for their ability to deliver hacyclovir and ganciclovir for extended periods of time following intravitreal injection to rabbits the drug concentrations attained in the vitreous and retina were high and steady for up to days unfortunately, these positive results were counteracted by the negative reaction observed in the lens opacification and in the aqueous humour turbidity with respect to the alteration of the normal physiological conditions of the eye, one of the problems that could be expected from the use of micro and nanospheres is their instability in the vitreous humor indeed, although no stability study has been reported, it could be accepted that, as in the case of other biological fluids, ie prevacid free powered by vbulletin lachrymal fluid, nanoparticles may suffer an aggregation process mediated by their interaction with proteins one of the alternatives to resolve this problem could be the peg coating approach described in the previous sections interestingly, while this approach has not been applied to plga nanoparticles yet, some evidence of its efficacy has been reported for peg coatedpolyhexadecyl cyanoacrylate nanoparticles these pegcoated nanoparticles, containing tamoxifen, have shown promising results for the treatment of experimental autoimmune uveitis, although no comparison was made between peg coated and noncoated nanoparticles therefore, and in spite of these promising results, the potential application of these nanoparticles will greatly depend on their tolerability and biodegradability in the ocular environment finally, nonpolymeric nanoparticles have also been reported for intraocular drug delivery concretely, prevacid free powered by vbulletin merodio et alsl evaluated the ocular toxicity induced by the prolonged presence of the ganciclovirloaded albumin nanoparticles after their intravitreal injection to rats these authors detected the presence of these systems in the vitreous cavity for up to two weeks after their intraocular injection in addition, according to the authors, the histological evaluation of these adjacent tissues revealed a good tolerance in summary, the reports of the potential of nanoparticles as intraocular drug delivery systems indicate that while their characteristics appear to be appealing for such application, further studies are necessary to assess important issues that include their stability and biodistribution in the intraocular cavity, as well as their biocompatibility and absence of toxic reactions or alterations of the normal function of the prevacid free powered by vbulletin eye conclusions and outlook despite extensive research in the field, the major problem in ocular drug delivery is the attainment of an optimal drug concentration at the intended site of action for a sufficient period of time the site of action maybe located on the eye surface or in the inner ocular structures the important barriers that need to be overcome in order to reach the target site limits not only the number of medications available for the treatment of ocular diseases, but also the extent to which those available can be used without incurring undesirable systemic side effects from the results described in this chapter, it is possible to conclude that nanoparticles offer great chances of solving these limitations, while still benefiting from prevacid free powered by vbulletin their topical administration as eye drops indeed, nanoparticles, depending on their composition, are significantly retained on the ocular mucosa, and from this location, they deliver the associated drugs for extended periods of time this situation normally results in an enhanced and prolonged therapeutic response, and also in a decrease in the side effects the results reported so far have also evidenced that both the extent of interaction and the penetration depth of the colloidal systems with the cornea, can be modulated by the selection of an appropriate coating in addition to these beneficial effects associated with the topical ocular administration, nanoparticles offer an interesting potential in terms of improving intraocular drug administration this potential includes not only the prolongation of the residence time prevacid free powered by vbulletin of drugs in the eye, but also their targeting to the retinal cells finally, significant efforts are currently underway to develop highly sophisticated nanoparticles functionalized with specific targeting ligands ie lectins and antibodies advances in this area are expected to open new avenues for the diagnostic and therapy including gene therapy of ocular disorders acknowledgments the authors would like to thank the spanish ministery of education and science for the financial support of some of the studies described in this chapter refs matc and safc references mitra ak mucoadhesive polymers in ophthalmic drug delivery systems, in mitra ak ed, ophthalmic drug delivery systems marcel dekker new york lang jc ocular drug delivery conventional ocular formulations adv drug del rev alonso mj and sanchez a prevacid free powered by vbulletin the potential of chitosan in ocular drug delivery j pharm pharmacol chrai ss and robinson jr ocular evaluation of methylcellulose vehicle in rabbits j pharm sci wood rw, lee vhk, kreuter j and robinson jr ocular disposition of polyhexyl cyanoacrylate 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nanoparticles in rabbit conjunctival epithelial cell layers pharm res calvo p, sanchez a, martinez j, lomez mi, calonge m, pastor jc and alonso mj polyester nanocapsules as new topical ocular delivery systems pharm res saettone mf, chetoni p, torracca mt, burgalassi s and giannaccini � evaluation of mucoadhesive properties and in vivo active of ophthalmic vehicles based on hyaluronic acid int} pharm saettone mf, giannaccini b, chetoni p, torracca mt and monti d evaluation of sodium hialuronate as adjuvants for topical ophthalmic vehicles int} pharm lutjendrecoll e, schenholm m, tamm e and tengblad a strattera with provigil visualization of hyaluronic acid in the anterior segment of rabbit and monkey prevacid free powered by vbulletin eyes exp eye res lehr cm, bowstra ja, schacht eh and junginger he in vitro evaluation of mucoadhesive properties of chitosan int ] pharm borchard g, lueben hl, de boer ga, coos verhoef j, lehr cm and junginger he the potential of mucoadhesive polymers in 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bioavailability of peg coated nanospheres } pharm sci calderone r and wadsworth e adherence molecules of candida albicans analysis of hostpathogen interactions implications for pathogenesis j microbiol methods kompella ub and lee vhl means to enhance penetration delivery systems for the enhancement of peptide and protein drugs design considerations adv drug del rev bishop pn, bonshek re, jones cpj, ridway aea and stoddart rw lectin binding sites in normal, scarred, and lattice dystrophy corneas br j ophthalmol nicholls tj, green kl, rogers dj, cook jd, wolowacz s and smart jd lectins in ocular drug delivery int} pharm norley sg, huang l and rouse �� targeting of drug prevacid free powered by vbulletin loaded immunoliposomes } immunol walsh g biopharmaceutical benchmarks nat biotechnol gref r, minamitake y, peracchia mt, trubetskoy v, torchilin v and langer r biodegradable longcirculating polymeric nanospheres science olivier jc, huertas r, lee hj, calon f and pardridge wm synthesis of 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control rel herrerovanrell r and refojo mf biodegradable microspheres for vitreoretinal drug delivery adv drug del rev moritera t, ogura y, yoshimura s, kuriyama s, honda t, tabata t and yoshito i feasibility of drug targeting to the retinal pigment epithelium curr eye res kimura h, ogura y, moritera t, honda, y, tabata y and ikada y in vitro phagocytosis prevacid free powered by vbulletin of polylactide microspheres by retinal pigment epithelial cells curr eye res bourges jl, gautier se, delie f, bejjani ra, jeanny jc, gurny r, benezra d and behar cohen ff ocular drug delivery targeting the retina invest ophthalmol vis sci bejjani ra, benezra d, cohen h, rieger j, andrieu c, jeanny jc, gollomb g and behar cohen ff nanoparticles for gene delivery mol vis elsamaligy m, ronjanasakul y, charlton jf, weinstei gw and lim jk ocular disposition of nanoencapsulated acyclovir and ganciclovir drug del kozak y, andrieux k, villarroya h, klein c, thillayegoldenberg b, naud mc, garcia e and couvreur p intraocular injection of tamoxifenloaded nanoparticles eur } immunol merodio m, irache jm, valamanesh f and mirshahi m ocular disposition and tolerance of ganciclovirloaded prevacid free powered by vbulletin albumin nanoparticles after intravitreal injection in rats biomaterials this page is intentionally left blank nanoparticles and microparticles as vaccine adjuvants janet r wendorf, manmohan singh and derek t ohagan introduction one of the most important current issues in vaccinology is the need for new adjuvants and delivery systems many of the vaccines currently in development are based on purified subunit proteins, recombinant molecules, synthetic peptides or plasmid dna unfortunately, it is clear that this new generation of vaccines will be less immunogenic than traditional vaccines, and will require better adjuvants and delivery systems to induce optimal immune responses, in addition, nonliving vaccines have generally proven ineffective at inducing potent cell mediated immunity cmi, particularly of the thl type t helper cells can be prevacid free powered by vbulletin classified into th and thl subtypes, mainly based on their cytokine production profile, with thl responses being characterized by the production of � interferon thl responses are likely to allow the development of vaccines against important infectious diseases, including hcv and hiv immunological adjuvants were originally described by ramon as substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone this broad definition encompasses a very wide range of materials however, despite extensive evaluation of a large number of candidates over many years, the only adjuvants currently approved by the us food and drug administration are aluminum based mineral salts, generically known as alum alum has a good safety record but comparative studies show that prevacid free powered by vbulletin it is a weak adjuvant for antibody induction to recombinant protein vaccines and induces a th, rather than a thl response in addition, alum is not effective in inducing mucosal iga antibody responses moreover, alum adjuvants can induce ige antibody responses and have been associated with allergic reactions in some subjects, although alum has been used as an adjuvant for many years, its mechanism of action remains poorly defined it was originally thought to provide a depot effect, resulting in the persistence of antigen at the injection site however, more recent studies involving radiolabeled antigens suggest that alum does not establish a depot at the injection site recent work has indicated that alum upregulates co stimulatory signals on human monocytes and promotes the prevacid free powered by vbulletin release of il alum adsorption may also contribute to a reduction in toxicity for some vaccines, due to the adsorption of contaminating lps a key issue in adjuvant development is toxicity, since safety concerns have restricted the development of adjuvants ever since alum was first introduced more than years ago many experimental adjuvants have advanced to clinical trials and some have demonstrated high potency, but most have proven too toxic for routine clinical use for standard prophylactic immunization in healthy individuals, only adjuvants that induce minimal adverse effects will prove acceptable additional practical issues that are important for adjuvant development include biodegradability, stability, ease of manufacture, cost and applicability to a wide range of vaccines adjuvants can be used to improve the immune prevacid free powered by vbulletin response to vaccine antigens in several different ways, including increasing the immunogenicity of weak antigens, enhancing the speed and duration of the immune response, modulating antibody avidity, specificity, isotype or subclass distribution, stimulating ctl, promoting the induction of mucosal immunity, enhancing immune responses in immunologically immature, or senescent individuals, decreasing the dose of antigen in the vaccine to reduce costs, or helping to overcome antigen competition in combination vaccines the mechanisms of most adjuvants still remains poorly understood, since immunization often activates a complex cascade of responses, and the principle mechanism of the adjuvant is often difficult to discern clearly however, if one accepts the geographical concept of immune reactivity, in which antigens that do not reach the local lymph nodes do not induce prevacid free powered by vbulletin responses, it becomes easier to propose mechanistic interpretations for some adjuvants, particularly those based on a delivery mechanism such as nanoparticles and microparticles if antigens which do not reach lymph nodes do not induce responses, then any adjuvant which enhances delivery of antigen into the cells that traffic to the lymph node may enhance the response a subset of dendritic cells dc are thought to be the key cells which circulate in peripheral tissues and act as sentinels, being responsible for the uptake of antigens and their transfer to lymph nodes, where they are then presented to t cells circulating immature dc are efficient for antigen uptake, while mature dc are efficient at antigen presentation to t cells hence, promoting antigen uptake into prevacid free powered by vbulletin dc, trafficking to lymph nodes and dc maturation are thought to be the key components to the generation of potent immune responses dc are thought to be the most effective antigen presenting cells apc, although macrophages can also function in this role it can be argued that the role of adjuvants for recombinant vaccines is to ensure that the vaccine resembles infection closely enough to initiate a potent immune response in addition, the innate immune system directs the balance of humoral and cmi, and adjuvants can control the type of acquired immune response induced adjuvants can be divided into different broad groups based on their principal modes of action, depending on whether or not they have direct immunostimulatory effects on apc, or whether they prevacid free powered by vbulletin function as antigen delivery systems particulate adjuvants eg emulsions, microparticles, iscoms, liposomes, virosomes and viruslike particles have comparable dimensions to the pathogens which the immune system evolved to combat immunostimulatory adjuvants may also be included in particulate delivery systems to enhance the level of response, or to focus the response through a desired pathway, eg thl in addition, formulating potent immunostimulatory adjuvants into delivery systems through restricting the systemic circulation of the adjuvant may limit adverse events nanoparticle and microparticle adjuvants generally act as delivery systems, although the materials they are made of may also have some adjuvant effect in the studies from onwards, kreuter et al, described the use of polymeric nanoparticles nm to nm in size as adjuvants for adsorbed and prevacid free powered by vbulletin entrapped vaccines however, the polymethyl methacrylates used in these studies are degraded in vivo only very slowly faster degrading particles prepared with the more biocompatible polylactic acid pla and poly lactidecoglycolide plg polymers have subsequently been extensively investigated as adjuvants size was shown to be an important parameter affecting the immunogenicity of microparticles, since smaller particles xm were significantly more immunogenic than larger ones, with plg particles of size xm mean of xm compared with xm mean of xm with entrapped staphylococcal enterotoxin b, the serum igg response was much higher with the smaller particles with a model antigen entrapped in pla particles, there was an increased antibody response with particles xm compared with particles with mean sizes larger than xm the effect prevacid free powered by vbulletin of particle size on immunogenicity is likely to be a consequence of enhanced uptake into lymphatics and greater uptake into antigen presenting cells for the smaller sized particles, since only microspheres �� were transported to the spleen the advantages of particles with a mean size of less than microns for optimal immune responses has been demonstrated on a number of occasions, but the data supporting the use of nanoparticles is less convincing in addition to the inherent immunogenicity of nano versus microparticles, carrier capacity and the efficiency of antigen entrapment in different formulations also needs careful consideration the dividing line between nanoparticles and microparticles is ill defined, with some sources considering nm particles to be nanoparticles, while the united states patent office has the prevacid free powered by vbulletin class definition for nanotechnology using the scale nm or slightly larger in this chapter, the evidence supporting the advantages of nanoparticles versus microparticles will be critically assessed previous reviews have focused on alternative delivery routes, including nasal and oral immunization in addition, the use of nanoparticles for dna vaccines, the stability of vaccines following microencapsulation, the cellular uptake of nanoparticles and microparticles, and the overall role of adjuvants in the immune response, has also been reviewed although a wide variety of microparticles and nanoparticles have been developed as possible vaccine adjuvants, we will focus primarily on systems where in vivo immune responses have been reported following systemic immunization, although mucosal immunization will be briefly covered nanoparticle and microparticle preparation methods there are many prevacid free powered by vbulletin biodegradable or biologically compatible polymers that have been used for the preparation of nanoparticles and microparticles different methods have been used for particle formation and these methods have optimal size ranges as well as suitability for use with different polymeric agents the preparation methods used, the typical size ranges, polymeric materials and antigens that have been evaluated are summarized in table nanoparticles and microparticles made from polyesters nanoparticles and microparticles made from poly lactidecoglycolide plg and poly lactide pla and their derivatives have been widely investigated for vaccine delivery using preformed polymers, particles with entrapped antigens can be prepared from a variety of emulsification evaporation methods these methods are based on the formation of a multiple emulsion water in oil in water emulsion from prevacid free powered by vbulletin which the oil phase, an organic solvent used to dissolve the polymer, is evaporated, resulting in the preparation of an aqueous suspension of particles this technique table summary of various particle preparation methods using different polymers and mixing conditions material method size range antigen type comments references plg or pla emulsification evaporation nm �� dna, protein small molecules smaller particles with increased surfactant concentration [ , ] plg or pla solvent displacement nm nm protein, small molecules smaller particles with lower polymer concentrations [] polycyanoacrylate chain polymerization nm nm protein concerns about residual monomers [, ] subramolecular crosslinking nm protein [, ] biovectors chitosan sodium sulfate precipitation nm � m protein [] chitosand complexation nm nm d complex forms spontanously with correct ratio [] prevacid free powered by vbulletin carbon nanotubes modification for covalent attachment nano protein, small molecules undetermined biological compatibility [] calcium phosphate various nm �� dna, protein [, ] generally has a lower limit for the particle preparation of about �� nm, although nm particles can be created with a low plg concentration and an increased surfactant concentration plg particles, with encapsulated model proteins having diameters as small as nm, can also be formed by sonicating the emulsions various alternative approaches have also been described based on emulsions, including spray drying and phase separation, however, one of the drawbacks of microencapsulation of antigens is the instability of the antigen due to the exposure to solvents and the high shear force during microparticle preparation, an alternate approach to encapsulation is prevacid free powered by vbulletin to adsorb the antigen onto the microparticles after the particle has been formed, avoiding the exposure of the antigen to solvents and high shear the solvent displacement method also sometimes referred to as interfacial deposition was first described by fessi et al, and allows the preparation of nanoparticles from preformed polymers a watermiscible solvent ie acetone is used to dissolve the polymer with magnetic stirring, which is then added to an aqueous solution the nanoparticles are formed by diffusion and the solvent is eliminated by evaporation depending on the solvent, polymer type, polymer concentration and addition of emulsifiers, these particles can range in size from nm to nm or larger, the antigens for encapsulation need to be water soluble and compatible with the watermiscible prevacid free powered by vbulletin solvent however, the encapsulation efficiency is often low for watersoluble molecules nanocapsules, with an internal oil core, can be formed when a small volume of oil is introduced into the organic phase, and these can be used to dissolve less watersoluble antigens, and to increase encapsulation efficiency however, one limitation of this approach is the preparation of low particle concentrations, due to the dilute initial polymer concentrations necessary in producing nm particles in addition to plg, other polymers used to prepare nanoparticles include polyecaprolactone pcl, and sulfobutylated polyvinyl alcoholgplg chain polymerization of modified cyanoacrylate monomers has also been used to make particles with a diameter of loonm, and with an additional polysaccharide coating, particles ranging in size from to nm can be produced prevacid free powered by vbulletin polymerization preparation of nanoparticles has also been used for methyl methacrylate polymers however, with the polymerization approach to nanoparticles preparation, there are concerns about the levels of residual monomers in the final formulation supramolecular biovectors smbv are positively charged particles with a polysaccharide core surrounded by a phospholipidic layer, with a mean size of nm , they are made from maltodextrins that were crosslinked with , epoxychloropropane and branched with glycidyl trimethylammonium to form a gel, which was then homogenized to give the nanoparticles, with the lipids added for the layer outside antigens were then adsorbed to the nanoparticles nanoparticles and microparticles made with chitosan chitosan is a natural biodegradable polymer of glucosamine and nacetylglucos amine it is made from the partial deacetylation of chitin prevacid free powered by vbulletin which is found in the shells of crustaceans it has been shown to be an effective adjuvant for the intranasal delivery of vaccines, enhancing tcell response and antibody levels when used as a soluble polymer chitosan is cationic and readily binds negatively charged materials, including dna and the sialic acid found on cell surfaces chitosan and chitosan coated particles can be made using several methods using a chitosan with a low molecular weight, nm chitosan particles can be formed by sodium sulfate precipitation with sonication, the particles can be reduced in size to nm chitosan coated polyecaprolactone particles were made using the solvent displacement method described above, with sizes ranging from nm with an emulsification method, chitosan was dissolved in the external phase to prevacid free powered by vbulletin form nm plg particles with a chitosan coating nanoparticles in the range of nm to nm can be formed spontaneously upon mixing of chitosan with dna the zeta potential surface charge can vary from negative to positive, depending on the ratio of dna to chitosan, although some particles were unstable and showed aggregation other nanoparticles and microparticles functionalized carbon nanotubes have been investigated as particles for vaccine delivery through organic modification, multiple sites for covalent attachment can be made available for small molecules, sugars, peptides or proteins the biological compatibility of carbon nanotubes is not certain yet, since they are not biodegradable another nanoparticle preparation method uses emulsifying wax of cetyl alcohol and polysorbate in combination with sds to create microemulsions, which are prevacid free powered by vbulletin then cooled to form particles ranging in size for to nm, depending on the sds concentration calcium phosphate particles have been studied for vaccine delivery, but are usually �� in size however, calcium phosphate nanoparticles have also been studied, although the reported mean size was xm, and these formulations were called nanoparticles alternative calcium phosphate particles of mean size nm have been used to encapsulate plasmid dna for gene therapy application adjuvant effect of nanoparticles and microparticles the adjuvant effect achieved as a consequence of the association of antigens with particles has been known for many years the enhanced immunogenicity of particulate antigens is unsurprising, since pathogens are particulates of similar dimensions and the immune system has evolved to deal with these particulate delivery prevacid free powered by vbulletin systems present multiple copies of antigens to the immune system and promote trapping and retention of antigens in the local lymph nodes moreover, particles are taken up by macrophages and dendritic cells, leading to enhanced antigen presentation and the release of cytokines, so as to promote the induction of an immune response antigen uptake by apc is enhanced by the association of antigen with polymeric particles, or by the use of polymers or proteins which self assemble into particles a particularly attractive feature of particles is their ability to control the rate of release of entrapped antigens many alternative antigen delivery systems that are available are particulates, including liposomes, iscoms, micelles and emulsions aluminum adjuvants have several limitations which has encouraged the search prevacid free powered by vbulletin for alternative approaches aluminium adjuvants are not effective for all antigens, induce some local reactions, induce ige antibody responses and generally fail to induce cellmediated immunity, particularly cytotoxic tcell responses in the early studies, microparticles with entrapped protein, and peptide antigens were shown to induce cytotoxic t lymphocyte ctl responses in mice following systemic and mucosal immunization microparticles also induced a delayed type hypersensitivity dth response, which is thought to be mediated by thl cells, and potent t cell proliferative responses the limited data available on the induction of cytokine responses in cells from animals immunized with microencapsulated antigens indicates that microparticles preferentially induce a thl type response, hence, microparticles may possess some inherent advantages over the more established alum based adjuvants macrophages prevacid free powered by vbulletin have been reported to be responsible for phagocytosis and the presentation of particulate antigens through the cytosolic mhc class i restricted pathway however, dendritic cells are also likely to play an important role in the presentation of particulate antigens and the release of cytokines to promote a thl type response the effect of particle size on antibody induction and cell mediated immunity has been investigated, and it has been concluded that zm particles are generally better than larger ones however, the data supporting the benefit of nanoparticles over microparticles is considerably less convincing nanoparticles and microparticles as mucosal adjuvants mucosal administration of vaccines offers a number of advantages over the traditional approach to vaccine delivery, which normally involves systemic injection using a needle and prevacid free powered by vbulletin syringe mucosal delivery would eliminate the possibility of infections caused by inadequately sterilized needles or the reuse of needles also, mucosal vaccines might result in the induction of mucosal immunity at the sites where many pathogens initially infect hosts mucosal delivery most commonly involves oral and intranasal in immunization, although alternate routes are also available the potency of particles for mucosal delivery is generally dependent on their ability to be taken up across the mucosal epithelium in many studies, the uptake of particles by the mucosal associated lymphoid tissues malts of the peyers patches in the gut and the malt of the respiratory tract have been demonstrated, albeit a very inefficient process moreover, there is good evidence that the composition of the particles prevacid free powered by vbulletin impacts efficiency of uptake, including evidence that the binding of plg microparticles to m cells of the peyers patches is less efficient than the binding of latex particles a number of alternative approaches have been evaluated for the mucosal delivery of vaccines using particulate carriers of various mean sizes chitosan particles of nm with associated tetanus toxoid tt were administered in in mice, and induced significantly higher serum igg responses compared with free antigen in addition, sulfobutylated poly vinyl alcoholgplg particles of mean size nm with adsorbed tt were administered orally and in, as well as enhanced serum iga and igg for the in antibodies, compared with soluble controls smbv nanoparticles with hepatitis � surface antigen administered in induced cytotoxic t lymphocyte ctl prevacid free powered by vbulletin responses and higher serum igg antibody responses than soluble protein calcium phosphate particles size pm were used for the mucosal delivery of a herpes simplex virus type antigen and they induced greater mucosal iga and igg, and systemic igg responses, compared with soluble antigen however, the calcium phosphate particles were sized before the final protein coating, so it is unclear what size the particles were when they were actually administered overall, although these various observations support the contention that particulate antigens are better than soluble antigens for mucosal delivery, all approaches appear to fall short of any likelihood of commercial development moreover, the rationale for preparing nanoparticles rather than the more established microparticles is not necessarily clear data from studies evaluating the effect of prevacid free powered by vbulletin particle size on the induction of mucosal immunity, following mucosal administration of vaccines, has offered conflicting outcomes, depending upon the specific polymeric system evaluated table in one study, with the model antigen ovalbumin ova adsorbed to chitosan particles of varying sizes nm, xm, xm for in administration, higher iga responses were seen with nm and xm particles, compared with the xm particles however, there was no difference in the response with the nm and the xm sized particles the antigen adsorption efficiency was similar for all particles, although no information is reported on the antigen release profile the conclusion that smaller nanoparticles were more immunogenic than larger microparticles was not confirmed by the paper table summary of various particles with different sizes showing prevacid free powered by vbulletin the mucosal adjuvant effect particle antigen sizes route result reference material chitosan model protein ovalbumin nm, ��, xm in comparable iga for smaller sizes, both greater than control im [] plg model protein bsa nm, nm, nm in igg responses of nm nm nm [] plg model protein bsa nm, nm, nm oral igg responses of nm nm nm [] pegpla protein tt nm, �� in comparable igg and iga responses for both sizes [] sulfobutylated protein nm oral igg and iga [] pvagplg tt nm, nm responses of nm nm, none for nm sulfobutylated protein nm in comparable iggiga [] pvagplg tt nm, nm response of nm, nm, none for nm chitosan protein tt nm in higher igg and iga compared to free prevacid free powered by vbulletin antigen [] smbv protein hbsag nm in high ctl and igg compared to free protein [] calcium protein nm mucosal higher igg and iga [] phosphate hsv compared to free antigen it is also known that particle charge is important for particles to be transferred to the apcs and the uptake by the malt this would suggest that positively charged chitosan particles may behave differently than negatively charged particles therefore, similar responses with micron and submicron particles may not apply to all particle types chitosan particles are biodegradable and may have an inherent immunostimulatory effect which pther polyesters plga lack thus, chitosan may be a promising candidate for a particulate system of size micron, although there is no justification for chitosan nanoparticles at prevacid free powered by vbulletin this moment another study evaluated the effect of particle size, using a specialized method of size nm with adsorbed tt administered orally and in this nanoparticle formulation induced increased serum iga and igg antibody responses in comparison with soluble antigen control kamm et al also examined , and nm particles following oral administration, the highest serum igg and iga responses were found with nm particles and the responses were progressively lower for the nm and nm nanoparticles following nasal administration, the nm and nm particles induced comparable igg and iga responses, which were higher than the responses with nm particles the authors speculated that the different size dependence observed for the different routes is due to the different translocation mechanisms in the nalt, as prevacid free powered by vbulletin compared with the galt the overall observations were that submicron nanoparticles were more immunogenic than larger particles the sbpvaplg may be preferred to pla for its faster degradation rate, however, the grafting chemistry required, renders it much less suitable for commercial development in contrast to the studies described above, it has also been claimed in some studies that xm particles are more effective than nanoparticles with bsa encapsulated into biodegradable nanospheres administered in and orally, nm plg particles elicited higher serum igg responses than nm and nm particles for in administration, the nm particles also induced higher serum igg responses than the nm particles the iggaiggl ratios with the different particle sizes were similar and higher than antigen alone and that with alum from prevacid free powered by vbulletin in vitro release studies, it was found that the nm particles did have a different release profile from the nm and nm particles, which may account for some of the differences seen in vivo for the oral studies, the authors hypothesized that nm and nm particles are more readily absorbed through the intestinal wall than nm particles, absorbed almost exclusively by peyers patch cells, leading to higher immune responses for the larger particles the two studies comparing particle size reached differing conclusions, but it is unclear whether this was due to the differences in polymers used for the studies, the different antigens used, or that of different formulations particle type as well as particle size can also have a strong influence on immune prevacid free powered by vbulletin responses using nm pegpla particles, nm pla particles and xm peg pla particles with encapsulated tt administered in, it was found that the nm and xm pegpla induced higher igg and iga antibody responses, compared with the pla particles although the two particle sizes were comparable, but the pegpla particles performed better than pla particles of the same size the peg pla particles are less hydrophobic than the pla particles this may explain some of the differences as particle uptake is strongly influenced by the hydrophobicity of the polymer the authors also hypothesized that the difference between the particle types is related to the propensity of the pla nanoparticles to aggregate in vitro, indicative of the relative stabilities of the particles in the prevacid free powered by vbulletin mucosal fluids although these particles are interesting, the pegpla polymer is not commercialy available, thereby hindering development the material of the particle system is an important factor to consider when comparing particles nanoparticles and microparticles as systemic adjuvants the use of nanoparticles as systemic adjuvants is summarized in table the advantage of particulates for vaccine delivery, compared with soluble antigens or alum, has been investigated in a number of studies and has been extensively evaluated by several groups , in one study, tt and cpg a known immunostimulatory oligonucleotide were coencapsulated within plg nanospheres of mean size nm and were evaluated in mice this formulation resulted in the induction of an enhanced antigen specific tcell proliferative response, in comparison with the soluble antigen plus cpg prevacid free powered by vbulletin the codelivery of tt and cpg within plg nanoparticles induced very strong serum igg response this is another instance of nanoparticles out performing soluble antigen however, the rationale for preparing nanoparticles rather than the more established microparticles is not necessarily clear table summary of various particles with different sizes showing the systemic adjuvant effect of these formulations particle antigen sizes route result reference material chitosan model protein ovalbumin nm, �� ip comparable igg for both sizes and greater than control [] plg model protein bsa nm, nm, nm sc igg responses of nm nm nm [] pla protein tt nm, �� im comparable titers, both sizes less effective than alum [] carbon peptide fmdv diameter ip more neutralizing [] nanotubes nm length nm prevacid free powered by vbulletin antibodies compared to free antigen plg protein tt cpg nm sc higher igg, thl type compared to free antigen enhanced tcell proliferation [] similarly, in another study, using an alternate carbon polymer, a nonbiodegradable, inorganic nanoscale particle was investigated carbon nanotubes with covalently linked peptides from a footandmouth disease virus were administered to mice ip and the carbon nanotubes elicited high levels of virusneutralizing antibodies, compared with the free peptide control carbon nanotubes introduce many problems they are not biodegradable and may be toxic hence they are not a good choice for vaccine formulation data from studies evaluating the effect of particle size on the induction of systemic immunity have offered conflicting outcomes a single dose of pla particles with tt encapsulated xm and prevacid free powered by vbulletin nm induced lower antitt titers than two doses of alum there were no significant difference between the larger and smaller particles the lower response with the particle formulation may be due to the limitation of having only one dose in particles, as compared with two doses on the alum the lower response with the pla formulation may also be due to the encapsulation process compromising of antigen stability the effect of particle size using bs a as an antigen was also evaluated by gutierro et aln the model antigen bsa was encapsulated into plg particles of varying sizes ie looonm, nm and nm and administered sc this elicited higher serum igg with the looonm, compared with the smaller particles the in vitro release prevacid free powered by vbulletin profile was different for looonm plg particles which may account for the differences in responses in another similar study, no size dependent effect was found with a model antigen ova adsorbed to chitosan particles of varying sizes nm, xm higher serum igg responses were seen with the particles compared with the soluble antigen, but there was no difference between the two sizes both studies described above used model antigens and the size conclusions may not be applicable beyond model antigens also, for some vaccines, antibody response may not directly correlate with protective efficacy of the vaccine one major difference between these two studies is the net surface charge of the particles it has been shown in vitro that positively charged particles polyllysine coated prevacid free powered by vbulletin polystyrene induced higher phagocytosis in dendritic cells surface charge as well as particle size might influence uptake by macrophages and dendritic cells therefore, the difference in size dependent behavior observed may also be more prominent involving particles of a specific charge recent studies have also shown that nanoparticles may exert an adjuvant effect for the induction of cell mediated immunity compared with soluble antigen, particle size may be an important parameter influencing the efficacy of microparticles as adjuvants for ctl induction nixon et al showed that microparticles nm induce better ctl responses than microparticles xm in another study, a nondegradable polystyrene nanoparticle with ova antigen covalently bound was administered id in mice it was seen that nm particles induced highest tcell responses in comparison prevacid free powered by vbulletin with the fxm size however, in this paper, the initial particle size is reported, but the size after covalent attachment of the antigen is not reported therefore, the conclusion cannot be confirmed by the findings reported delivery of dna using nanoparticles and microparticles the previous sections were mainly focused on the delivery of proteins or peptides, but dna nanoparticles and microparticles have also been used nanoparticle studies with dna as adjuvants are summarized in table dna plasmids are weakly immunogenic and particles may help boost the immune response cationic plg particles with adsorbed ���� of sizes nm, im, and zm were used to adsorb an hiv dna plasmid and delivered im the nm and xm particles induced, significantly enhanced igg titers, compared with prevacid free powered by vbulletin naked dna and the xm particles the �� particles were capable of inducing potent ctl responses, whereas naked dna failed to induce ctl activity it appeared in this study that the nm particles were better than the zm particles an additional study with more animals confirmed that the im and nm plg particles with adsorbed plasmid dna, induced comparable igg serum titers fig there was no advantage for the smaller sized particles it is believed that the efficient delivery of dna to apcs is table various particle based delivery systems for dna showing the mucosal systemic adjuvant effect of these formulations particle antigen sizes route result reference material plg ���� dna hiv nm, ��, �m im higher igg for nm, jxm compared to free prevacid free powered by vbulletin d [] chitosan dna peanut allergen nm oral higher iga and igga response compared to free d [] chitosan d tuberculosis nm pulmonary increased ifny compared to dna alone of im administration [] polylysineg dna hiv nm id higher igm, igg [] imidazoleatic env iga compared to acid free antigen nm im soluble nm xm soluble �� �� �� �� �� �� fig serum igg titers in groups � = for particles, n = for soluble of balbc mice immunized with either plgctabp gag dna of size nm or in or dna alone at two dose levels of xg and xg antibody titers are geometric mean titers � se at weeks postsecond immunization week time point after immunizations at day and day the prevacid free powered by vbulletin response from the nm and xm particles at both dose levels are not significantly different an important component of the adjuvant effect, since larger microparticles xm did not elicit a strong immune response the association of dna with nanoparticles and microparticles has been shown to be more effective than naked dna chitosandna particles nm of peanut allergen gene delivered orally, produced secretory iga and serum igga, compared with no detectable response with naked dna the pulmonary delivery of chitosandna particles average size of nm with plasmid dna encoding tcell epitopes from mycobacterium tuberculosis, were able to induce the maturation of dendritic cells and the increased level of ifny secretion, compared with the plasmid in solution or im delivery in another dna study, polylysinegraft prevacid free powered by vbulletin imidazoleacetic acid complexed with dna with a diameter of nm was used for the hiv env plasmid it was found that igg, igm and iga responses were increased several folds, compared with naked dna it was also speculated that this formulation may also help protect the dna from nuclease degradation based on the evidence in the literature, the rationale for preparing nanoparticles rather than the more established microparticles is not necessarily clear with the dna formulations conclusions a number of systems with different types of antigen proteins, peptides, dna have been investigated with the particles ranging in size from nm to nm for most systems, the critical particle size is zm, with particles in the range of nm to zm, often inducing comparable immune prevacid free powered by vbulletin responses in some cases, depending on the route of delivery, there may be increased immune responses with the smaller nanoparticles for in administration, there was no evidence that nm particles were better than micron particles for oral administration, some studies found enhanced responses with nanoparticles, compared with microparticles, while other studies found equivalence, or that microparticles elicited higher responses overall, the evidence for nanoparticles nm outperforming microparticles zm for enhanced immunogenicity is weak further examination is needed to support nanoparticles as a better formulation in place of microparticles also, some of the studies carried out were done model antigens and the same results maymay not apply to relevant antigens where vaccine efficacy is determined however, there are some advantages to nanoparticles compared with prevacid free powered by vbulletin microparticles that have not been directly addressed for instance, smaller nanoparticles sub nm can be sterile filtered, allowing the particle preparation to be a nonsterile process with terminal sterilization the distinction between nanoparticles and microparticles is usually made by the authors and there is no consistency in what constitutes a nanoparticle, and this needs careful consideration when comparing results from the literature the important size measurement point is immediately prior to administration, post lyophilization, or other processing, and this is not always reported more relevant endpoints such as protective efficacy may be crucial in distinguishing between nanoparticles and microparticles nanoparticles and microparticles both constitute a very effective vaccine delivery system some of these formulations are currently in preclinical and clinical evaluations acknowledgments we would like to acknowledge the contributions of our colleagues in chiron corporation to the ideas contained in the chapter, particularly, all the members of the vaccine adjuvants and delivery group references gupta rk and siber gr adjuvants for human vaccinescurrent status, problems and future prospects vaccine gupta rk, griffin p, jr, chang ac, rivera r, anderson r, rost b, cecchini d, nicholson m and siber gr the role of adjuvants and delivery systems in modulation of immune response to vaccines adv exp med biol ramon g sur la toxine et surranatoxine diphtheriques ann inst pasteur vogel fr and powell mf a compendium of vaccine adjuvants and 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