Review Article Recent Developments in Liposome-Based ...downloads.hindawi.com/archive/2013/167521.pdf · review depicts the current signi cance and future directions of liposome-based

Hindawi Publishing CorporationISRN Veterinary ScienceVolume 2013 Article ID 167521 8 pageshttpdxdoiorg1011552013167521

Review ArticleRecent Developments in Liposome-BasedVeterinary Therapeutics

Hassan Sadozai1 and Dorsa Saeidi2

1 Biomedical Sciences 218 Queenrsquos Quay West Toronto ON Canada M5J2Y62Animal Biology 7454 Conservation Road Guelph ON Canada N1H6J2

Correspondence should be addressed to Hassan Sadozai hassanasadozaigmailcom

Received 7 August 2013 Accepted 11 September 2013

Academic Editors Y-F Chang and M H Kogut

Copyright copy 2013 H Sadozai and D Saeidi This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Recent advances in nanomedicine have been studied in the veterinary field and have found a wide variety of applications Thepast decade has witnessed a massive surge of research interest in liposomes for delivery of therapeutic substances in animalsLiposomes are nanosized phospholipid vesicles that can serve as delivery platforms for a wide range of substances Liposomesare easily formulated highly modifiable and easily administered delivery platforms They are biodegradable and nontoxic andhave long in vivo circulation time This review focuses on recent and ongoing research that may have relevance for veterinarymedicine By examining the recent developments in liposome-based therapeutics in animal cancers vaccines and analgesia thisreview depicts the current significance and future directions of liposome-based delivery in veterinary medicine

1 Introduction

The veterinary pharmaceutical industry provides pharmaco-logical agents for a wide variety of farm companion andlaboratory animals Typically the optimal products must becost-effective safe easily administered [1] demonstrate invivo efficacy be nontoxic and display favourable pharma-cokinetics [2] The final factor is the most salient as 90 ofpotential therapeutic agents have low bioavailability and poorpharmacokinetics [2] In order to provide better therapeuticefficacy the pharmacological agents can be incorporated intonovel drug delivery systems [2 3]

Recent advances in nanotechnology have allowed for thedevelopment of novel nanodrug delivery systems such aspolymeric nanoparticles magnetic nanoparticles nanocrys-tals nanoemulsions and liposomes [2 3] These nanodrugdelivery systems are known to enhance the therapeutic indi-ces of the incorporated drugs through a number of waysThese delivery systems protect the entrapped agent from theinternal body environment improve the bioavailability andpharmacokinetics of the drug are able to evade immunecapture allowing for sustained-release of the drug over time[2 3] and lower drug-associated toxicity by improving

site-specific delivery [2] In light of the possibilities offeredby nanodrug delivery systems their therapeutic applicationshave been investigated and this area has fostered considerableveterinary research interest The term widely used to referto this novel area of research for both human and animalapplications is ldquonanomedicinerdquo [2ndash4]

Among the wide variety of existing drug-delivery sys-tems several liposome-based therapeutic agents in animalshave been evaluated over the past decade and have beendemonstrated to be highly versatile and easy to modify andare relatively simple to formulate [4 5] They are spheri-cal self-closed vesicles formed by one or more concentriclipid bilayers around an aqueous inner compartment withtherapeutic agents capable of being encapsulated within theaqueous cavity or the lipid bilayers of the liposomes [5]

The focus of this review will be to highlight recent devel-opments in liposome-based therapeutics that are relevantfor veterinary medicine This review will recap recent andongoing research on liposome-based therapeutics in cancertherapy vaccine delivery and pain management in speciesof veterinary and agricultural relevance This paper aimsto demonstrate the significance current relevance and thefuture potential of liposomes as nanosized delivery platforms

2 ISRN Veterinary Science

Table 1 An overview of the morphological characteristics of different types of liposomes [5]

Multilamellar vesicles Consist of several concentric bilayers Range in size from 500 to 5000 nm Ideal for trappinghydrophobic drugs in additional lamellae

Large unilamellar vesicles Consist of one concentric lipid bilayer surrounding a large inner aqueous environment Range in sizefrom 200 to 800 nm Ideal for trapping large amounts of hydrophilic drugs

Small unilamellar vesicles Consist of one concentric bilayer Small size in the range of 100 nm Ideal for long-term circulation

Hydrophilic drug

Hydrophobic drug

PEG

Targeting ligand

Cationic lipid

Phospholipid

Figure 1 A graphical depiction of the versatility of liposomes asdelivery platforms (lowastPEG poly-ethylene glycol)

in veterinary medicine Furthermore nanoparticles devel-oped for and tested in veterinary species may be relevantfor translation to human medicine In fact the pharmacoki-netic and toxicity profiles of nanoparticle formulations areoften tested in canine models [6] Hence liposome-basedtherapeutics that are relevant for veterinary species but alsohave relevance for human nanodrug development will bediscussed Due to the versatile applications of liposomesa review of recent developments in the field is warrantedespecially as it pertains to veterinary applications

2 Liposomes as Delivery Platforms

Liposomes were first described in the 1960rsquos by Alec Bang-ham who reported the ability of phospholipids to formclosed vesicles encircled by lipid bilayers that resemble cellmembranes (Figure 1) [5] The basic structure of liposomesinvolves the hydrophilic head groups of the lipid bilayerdirected towards the aqueous phases whereas the hydropho-bic tail groups are directed towards each other to form the

membrane core [5 7] Generally hydrophobic substances canbe entrapped within the lipid bilayer and hydrophilic sub-stances within the inner aqueous compartment [7] Alteringthe preparation parameters can yield vesicles with differentmorphological characteristics that are shown in Table 1

Liposomes serve as effective delivery platforms due toseveral favourable characteristics (Figure 1) They can encap-sulate both hydrophobic and hydrophilic compounds andcan be used for intracellular drug delivery [7] Moreoverthe vesicle size surface charge and surface properties canbe easily modified using different compounds and prepa-ration parameters [7 8] For example adding polymerssuch as poly(ethylene) glycol (PEG) to the liposomal surface(PEGylation) can create long-circulating liposomes that canevade capture from the reticuloendothelial system (RES)stay in the body longer and demonstrate extended-releaseof the encapsulated drug over time [9] Moreover attachingantibodies and other markers to liposome surfaces canallow for diagnostic imaging and targeted therapy [5 8]Finally liposomes can be designed for triggered release usingexternal stimuli such as pH ultrasound and temperature[5 10] Temperature-sensitive liposomes are designed withthermosensitive polymers that have lower critical solutiontemperatures (LCST) attached to their surface [10] At tem-peratures below their LCST (usually 20∘C) the polymerchains are stable and hydrated but at temperatures higherthan the LCST (at around 39ndash42∘C) they become dehydratedand disrupt the lipid bilayer resulting in an immediate releaseof entrapped contents (Figure 2) [10] The aforementionedcharacteristics of liposomes demonstrate their potential inseveral areas of veterinary medicine In particular liposomescan serve as potent delivery platforms for cancer therapeuticsvaccine and analgesic drugs

21 Liposome-Based Cancer Therapeutics The rationale fornanoparticle based cancer therapeutics has been extensivelyreviewed [11ndash13] Modern cancer therapy involves the use ofseveral antineoplastic agents many of which are chemother-apeutic drugs These drugs are potent at eliminating cancercells in vitro but are observed to have significant barriersto in vivo efficacy [13] These barriers include a lack ofselectivity for cancer cells low bioavailability at tumour siteslarger volumes of distribution and toxicity to normal tissues[12] Nanotechnology-based drug delivery systems such asliposomes can overcome these barriers through a variety ofmechanisms Due to their small size (10ndash100 nm) they areideal for intracellular uptake have high encapsulation capac-ities and can be designed for specific targeting of tumourcells [12 13] Furthermore the intrinsic characteristics oftumour tissue such as leaky microvasculature and highly

ISRN Veterinary Science 3

ΔT

Thermosensitive (TS) polymerDenatured TS polymerDrug (doxorubicin)

Figure 2 Thermosensitive liposomes are potent sustained deliveryvehicles that can be triggered to release contents when desired

impaired lymphatic drainage can allow for accumulation ofthese nanoparticles within the tumour [13]

Liposomes have demonstrated a promising potential fordelivery of anticancer drugs in animals Ranging as farback as 1995 clinical trials in dogs with canine splenichemangiosarcoma (HSA) demonstrated the enhanced anti-tumour potential of liposome-encapsulated muramyl tripep-tide [14] Liposome-encapsulated muramyl tripeptide conju-gated with phosphatidylethanolamine was given to dogs asan immunotherapy adjuvant to Doxorubicin chemotherapyand resulted in prolonged disease-free survival in the morbidcanines [14] Since then liposome-based cancer therapeuticshave shown encouraging results in animals with profoundimplications for veterinary oncology as well as human cancertherapyThat is the casewith liposome-encapsulatedDoxoru-bicin which demonstrates favourable pharmacokinetic pro-files and lower cardiotoxicity in human patients as opposedto free Doxorubicin [15] PEGylated liposomes containingDoxorubicin are available for clinical use in humans as Doxil(Caelyx in Europe) [15] Despite observable increases of druglevels at tumour sites the clinical outcomes of humanpatientstreated with liposome-encapsulated Doxorubicin have beenthe same as those treated with free Doxorubicin [15 16]The low response rate of these liposomal formulations waspurported to be due to a lack of understanding of drug releasefrom the liposomes [17]

Liposomes also serve as ideal vehicles for triggered rele-ase with external stimuli such as pH and temperature actingas the trigger (Figure 2) [5 10] A pilot study conducted indogs described the results from a phase I clinical trial ofDoxorubicin encapsulated within low-temperature sensitiveliposomes (LTSL) [16] LTSL administered to solid tumourswith simultaneous induction of tumour hyperthermia resultsin triggered release of 100 of their contents within 20seconds of achieving the transition temperature of 413∘C 18privately owned dogs with sarcomas and 3 with carcinomaswere recruited into the study Of the 21 dogs enrolled in thetrial 20 received two or more doses of the LTSL formu-lation and of these 12 had stable disease (lt50 decreasein tumour volume) and 6 had partial response to disease(gt50 and lt100 decrease in tumour volume) [16]This trial

demonstrated a novel approach to liposome-based drug deli-very to tumours

Use of liposomal formulations in conjunction with othertherapies as a multifaceted approach to veterinary oncologyhas also been investigated Due to liposome-based drugshaving longer in vivo circulations sensitizing agents can beloaded into liposomes to serve as potent pretreatment sensi-tizers for radiotherapy in cancer A study conducted in 2010demonstrated improved therapeutic outcomes in cats withadvanced feline soft tissue sarcomas when given liposomalDoxorubicin concomitantlywith daily palliative radiotherapy[17] Liposomal Doxorubicin has been shown to sensitizetumour cells to concomitantly administered radiotherapy[17] Despite the small sample size (119899 = 10) the results wereencouraging with 7 cats achieving partial (119899 = 5) or complete(119899 = 2) response for a duration of 237 days [17] In addition toDoxorubicin other antineoplastic agents have also been stud-ied as liposome-encapsulated formulations In a 2010 study itwas demonstrated that liposome-encapsulated clodronate abisphosphonate drug could be utilized for malignant histio-cytosis therapy in dogs [18] Malignant histiocytosis (MH) isan aggressive malignancy of the myeloid lineage in dogs andis resistant to many conventional chemotherapeutic drugs[17] The liposome-encapsulated clodronate was observed toeffectively kill MH cells in vitro and was subsequently testedin 5 dogs with MH The dogs were given 2 IV treatmentsof 05mLkg liposomal clodronate administered 2 weeksapart resulting in significant tumour volume reduction in2 out of the 5 animals enrolled in the treatment [18] Akey weakness of recent investigations using liposome-basedcancer therapeutics is the small number of animals beingtested In order to justify further development of a specificformulation by the veterinary pharmaceutical industry theproduct will require large multicenter trials analogous tothose conducted in human medicine

In addition to chemotherapeutic substances liposomeshave also been evaluated as DNA delivery vectors for genetherapy of cancer In particular cationic liposomes (CLs)have been demonstrated as promising candidates for genedelivery [19ndash21] Cationic liposomes are composed of cationicand ldquozwitterionicrdquo helper lipids that can form stable com-plexes with polyanionic DNA (liposome-DNA complexes orlipoplexes) [20 21] ldquoLipofectionrdquo or liposome-based DNAtransfection shows 100 DNA entrapment and can theo-retically offer a valid alternative to viral gene delivery forcancer therapy [19 20] Viral gene delivery displays strongtransfection capacity but suffers from several in vivo barriersto efficacy such as toxicity immunogenicity inability tomaintain high levels of gene expression and an inability topersist in targeted cells [20 22] Unfortunately lipofectionsuffers from low transfection efficiency compared to that ofviral vectors and this impedes their broad application asnonviral alternatives for gene delivery [19 20] Hence muchresearch is currently being conducted to understand thestructural interactions of these CLs with DNA as well as withintracellular components [20] Notwithstanding the afore-mentioned limitations liposome-DNA complexes (LDCs)offer a highlymodifiable nontoxic platform forDNAdeliveryto humans and animals [19 21] A pilot study conducted in


2007 investigated the use of these LDCs as effective cancervaccine adjuvants in dogs [23] LDCs were used to constructa vaccine consisting of the cell lysates from canine allogeneichemangiosarcoma (HSA) cell lines which was coadmi-nistered along with Doxorubicin to 28 dogs with HSA [23]The dogs mounted a strong antibody response to canine HSAcells and of 28 dogs receiving the joint therapy 13 demon-strated increased overall median survival time [23] LDCshave also been evaluated for delivery of endostatin DNAa VEGF antagonist for antiangiogenic therapy of cancer indogs with cutaneous soft-tissue sarcomas [24] The studydid not observe detectable levels of endostatin gene expres-sion but a significant response in tumour physiology wasobserved Out of 13 dogs treated with 6 weekly intravenousinfusions of LDCrsquos 8 had stable disease Moreover in 6 of 12dogs that received complete treatment tumour microvesseldensity was significantly decreased due to an antitumourimmune response mediated by tumour-infiltrating lympho-cytes (TILs) and purported to have been elicited by theliposomes [24] These studies demonstrate that liposome-based gene delivery warrants further investigation for animalcancers particularly in light of the safety issues associatedwith viral gene delivery [22] If proven effective liposomescan serve as potent platforms for gene therapy of cancer aswell as eliciting antitumoural immune response Finally it isimportant to note that recent developments in nanoparticle-based cancer therapeutics are aimed towards nanoparticleswith high specificity for certain cells and furthermore certainorganelles within a cell [25] A recent study reported the useof a Doxorubicin-containing liposomes conjugated with a10 amino acid ldquotumour metastasis targetingrdquo (TMT) peptide[26] The TMT liposomes were found to be actively targetedto and endocytosed by metastatic tumour cells in a nudemouse animal model The active-targeted liposome formu-lation of Doxorubicin demonstrated effective inhibition ofmetastatic tumours in vivo with minimal side effects [26]This study demonstrated the effectiveness of actively targetedcancer therapeutics These liposome-based cancer therapeu-tics promise improved animal welfare increased productivityin farm animals and finally translational tools for humanmedicine after proven efficacy in animals

22 Liposomes for Delivery of Vaccines In recent years lipo-somes have been evaluated as platforms for vaccine design[25] In particular food safety concerns and zoonotic diseasecontrol necessitate further research into vaccines for foodanimal species [27] Vaccines are predicated on the deliveryof inactivated pathogens to invoke a potent lasting responsein the host [25] In recent years there has been a drive todevelop safer recombinant proteins and synthetic peptides asldquosubunitrdquo vaccines [28] However these vaccines often havepoor immunogenicity and like other vaccines require potentadjuvants to improve host immune response [29] Thereforethere has been considerable research on the use of nanosizedbased delivery systems such as liposomes for deliveringadjuvants that can enhance the immunogenicity of novelvaccines [28 29] These systems can potentially enhanceimmunogenicity through a number of ways First manynanoparticles can mimic pathogen-associated molecular

patterns activating innate immune response through patte-rn-recognition receptors [29] Second nanoparticles such asliposomes are taken up preferentially by antigen presentingcells resulting in an enhanced T-cell activation [30] Inparticular cationic liposomes serve as potent vaccine designplatforms due to their ability to bind with DNA and elicit animmune response [20 31] Furthermore some nanoparticlescan be constructed with viruslike particles on their surfacethereby providing the necessary immune stimulationwithoutthe actual virus DNA that can cause infection [28] Finallydelivery systems such as liposomes can act as targetable depotformulations that provide extended delivery of antigen toa specific location for a designated amount of time [30]Due to the potentially favourable characteristics of liposomesfor vaccinations against a range of veterinary pathogensliposome-based vaccination in food animals has generatedmuch research interest in the past decade

In a study conducted in 2002 the viability of lipo-somes as vectors for ldquosubunitrdquo vaccines was demonstrated inpoultry [32] This study looked at vaccination with liposo-me-associated fimbriae antigens (SEF14 and SEF21) of thebacteria Salmonella enterica serovar Enteritidis a commonpathogen in animals and humans [32 33] Infection inhumans is usually associated with the ingestion of contam-inated chicken eggs egg products or chicken meat [34]Intraocular immunization with liposome-associated fimbrialantigens resulted in significant increases in IgA and IgG pro-files along with counts of antibody-producing lymphocytes[32] When subsequently challenged with live Salmonellaenteritidis the immunized group demonstrated significantlyless excretion of the bacteria in feces and nearly a 95inhibition of S enteritidis colonization in the caecum ascompared to the unimmunized control group [32] Since fecalexcretion of enteropathogens is one of the primary causes ofegg contamination this study also has implications for foodsafety and human health [32]

From the perspective of food safety and residue avoid-ance liposomes have also been evaluated for nonparenteralroutes of vaccine administration in food animals [34] Aviancolibacillosis is an acute problem in the poultry indus-try resulting in septicaemia and respiratory problems inboth broiler and layer breeds of poultry [34] Hence astudy conducted in 2009 investigated the nonparenteraladministration of liposome-encapsulated inactivated APEC(avian pathogenic E coli) as a vaccine for control of aviancolibacillosis [34] The inoculated chickens produced IgAand IgG antibodies in their oral mucus When subsequentlychallenged with a live strain of APEC the immunizedchickens were found to have lower bacterial counts in theblood and no serious adverse effects as a result of inoculation[34] This study was the first to demonstrate the induction ofmucosal immunity in poultry using liposome-based vaccinesThe success of inducing immunity through nonoral routes ofadministration can be potentially translated for the vaccina-tion of other animals where drug and vaccine residues are animportant consideration for food safety

There is also evidence that nanoparticle-based vac-cine formulations for some diseases may demonstratehigher efficacy than commercially available formulations as


demonstrated by a chitosan-based nanoparticle vaccine forNewcastle disease (ND) [35] Similar improvement in efficacywas also shown in a recent study using a liposome-coatedversion of the commercial live ND vaccine [36] Newcastledisease caused by the ND virus (NDV) or avian paramyx-ovirus type 1 (APMV-1) is considered to be the most devas-tating poultry disease after highly pathogenic avian influenza(H5N1) and is endemic to many areas [36 37] Differentstrains of NDV result in a vast range of symptoms includingsudden death [36] Hence NDV vaccination is an importantconsideration in all poultry production units The aforemen-tioned study examined the differences in immune responsebetween chickens given liposome-encapsulatedNDVvaccineor the La Sota vaccine [36] The La Sota vaccine containsthe lentogenic live La Sota strain of the ND virus and canbe administered intranasally [35] The vaccine groups werevaccinated orally at 3 and 6 weeks of age and subsequentlychallenged with the virus The antibody production andcell counts were significantly higher in the birds vaccinatedwith the liposomal ND [36] After the second vaccinationat 6 weeks of age the antibody titre was also significantlyhigher for the liposomal-ND vaccine group than the LaSota vaccine group Some of the reasons why liposomal ND-vaccine performed better than the commercial vaccine arethat the types of liposomes used in this study were cationicliposomes which can fuse with cell membranes and that theycan evade capture due to their small individual particle size(under 100 nm) Therefore the liposome-based ND vaccinewas believed to have longer contact and better targeting tothe cells of the immune system [36]

Finally liposomes have also been used to design vaccinesagainst parasites in agricultural animals A novel investiga-tion demonstrated that liposome-DNA complexes carrying aplasmid encoding formicronemeMIC3protein resulted in aneffective immune response against this important parasite insheep [38] T gondii is a protozoan parasite found worldwideand is one of themost common causes of ovine abortion [38]Currently a live vaccine Toxovax is being used to protectagainst the parasite in sheep and there is a drive towardscreating a safer synthetic ldquosubunitrdquo vaccine for farm animalsand humans [39] The microneme MIC3 is an important celladhesion protein utilized by T gondii to enter host cells Aplasmid coding for the mature form of this protein was usedto create a liposomal DNA vaccine that was tested in a studysample of 36 two-year-old ewes It was demonstrated thatliposome-based vaccines also elicit strong immune responsesagainst parasitic pathogens and thuswarrant further study forvaccine design in livestock [39] The studies described abovedemonstrate that liposome-based vaccines have effectivelybeen tested against a diverse group of pathogens Henceliposomes can serve as platforms for vaccine delivery toboth food animals and companion animals Finally if cost-effective and mass produced vaccines for many food animalpathogens become available the lessons learnt from thesetrials would better inform the development of liposome-based vaccines against many human pathogens

23 Liposome-Based Analgesia The management of acuteand chronic pain is an important part of veterinary medicine

for laboratory animals domestic pets and farm animals [4041] However most pharmacological agents with analgesicproperties have a high volume of distribution and relativelysystemic half-life [40] In contrast with human medicinewhere for the most part patients can self-administer painmedications orally veterinary pain management requiresfrequent dosing and rigorous administration protocols [40]This necessitates frequent handling and higher logistical costsand increases risks of zoonotic infections for animal handlers[40 41] To overcome these obstacles novel drug deliverysystems are continually being devised [41 42] Liposomeshave been demonstrated to act as depot formulations for painmedication as far back as 1997 in a rat study on the would-infiltration capacity of liposomal bupivacaine [43] How-ever only in the past decade have several research groupsbegun to study the pharmacokinetic and pharmacodynamicsof liposome-encapsulated analgesics in various veterinaryspecies

Technological advances in recent years have made itpossible for the incorporation of many different types ofanalgesics into liposomes Some companies have also devisedproprietary formulations such as Depofoam bupivacainewhich consists of a single dose (15mgmL) of an extended-release liposomal injection of bupivacaine [44]This formula-tion has been evaluated in both rabbits and dogs and has beendemonstrated to provide extended-release analgesia with noadverse effects [44] Opioids remain the most widely studiedanalgesic drugs for liposomal delivery [42ndash45] The abilityof liposome-encapsulated oxymorphone (LE-oxymorphone)and liposome-encapsulated-hydromorphone (LE-Hydro) toprevent hyperalgesia in rat models of induced neuropathicpain has beenwell documented [9 44] In fact LE-Hydrowasdemonstrated to prevent hyperalgesia for as long as 5 daysafter administration in rats [40] A recent investigation thatstudied artificially induced pain models in green cheeked-conures (Pyrrhura molinae) demonstrated that liposome-encapsulated butorphanol tartrate provided extended releaseanalgesia for alleviation of this pain [45] In order to evaluateliposomes for analgesic delivery at a broader veterinary scaleit would be essential to conduct studies in larger animalssuch as dogs to gauge behavioural and pharmacodynamicsresponses A pharmacodynamics study conducted in 2011examined the side-effects of LE-Hydro in healthy beaglesfollowed by a determination of analgesic efficacy of LE-Hydroin other dogs undergoing ovariohysterectomies (OVH) in thesame hospital [46] The LE-Hydro was well-tolerated withrespiratory depression being the most common effect [46]This study was crucial in establishing that liposomes can actas nontoxic sustained-release formulations for opioids

Despite the fact that much of the research in liposome-based analgesics has focused on encapsulating opioids nons-teroidal anti-inflammatory drugs or NSAIDs for short havealso been evaluated These are inhibitors of the enzymescyclooxygenase (COX)-1 and COX-2 [47] For example arecent study evaluated the use of diclofenac liposomal creamfor experimentally induced osteoarthritis in horses Twenty-four healthy horses aged 2ndash5 years old were selected for thisstudy After osteoarthritis was artificially induced they weredivided into three groups of 8 receiving no treatment oral


Table 2 An overview of some of the liposome-based therapeutic systems studied in recent years with clinical significance for veterinarymedicine (For explanation of symbols please refer to legend)

Species Agent Diseasecondition ReferenceDogs Doxorubicin (thermosensitive liposomes) Spontaneous canine tumours [16]◼

Cats Doxorubicin in conjunction with radiotherapy Soft-tissue sarcoma [17]Dogs HSA cell lysates Canine hemangiosarcoma (HSA) [23]998771

Dogs Endostatin DNA Soft-tissue sarcoma [24]998771

Chickens Salmonella fimbriae proteins Salmonella enterica vaccine [32]Chickens Inactivated APEC (avian pathogenic E coli) Avian colibacillosis vaccine [34]998771

Chickens Newcastle disease virus Newcastle disease vaccine [35]998771

Sheep MIC3 protein from T gondii Toxoplasma gondii vaccine [39]998771

Green-cheeked conures Butorphanol tartrate Experimentally induced arthritic pain [45]Dogs Hydromorphone Postoperative pain [46]Horses Diclofenac Osteoarthritis pain [48]◼Clinical trial 998771Pilot study or primary evaluation in listed species

administration of the NSAID phenylbutazone and topicalapplication of diclofenac liposomal cream (DLC) respec-tively [48] 73 g of DLC was topically applied to the affectedarea twice a day and was observed to significantly modifyclinical signs of lameness in the affected limb and displayno treatment-related detrimental effects Furthermore DLCwas observed to induce far less carpal bone sclerosis andoverall cartilage erosion as compared to phenylbutazone [47]Actually DLC is now successfully marketed in the US asa liposomal cream for osteoarthritis pain management inhorses [48] The fact that liposomes perform well both assystemic administrations and as topical applications warrantstheir further evaluation and indicates their continued clinicalsignificance for pain management in veterinary medicineControlled release formulations for analgesic drugs offer adual advantage for biomedical research They permit theadequate pain management in various companion and exoticanimal species and therefore allow these species to be used asmodels for human nanomedicine

3 Conclusions and Future DirectionsAs with all nanoparticles future consideration for use war-rants consideration of toxic effects in an animal or humanbody Despite the fact that liposomes are nontoxic liposomeslipid micelles and solid-lipid nanoparticles are known tocause acute hypersensitivity reactions (HSRs) [49] Thesereactions are putatively caused by the activation of thecomplement (C) activation by the surface of the lipid particlesand can be studied in animal sensitivity models [49] In apig sensitivity model the most commonly observed adverseeffects were shown to be anaphylactoid shock characterizedby pulmonary hypotension and cardiac arrhythmias [49]Therefore few liposome-based therapeutics are currentlyavailable commercially for human and animal medicine[4 15] Further trials in large-scale animal studies will berequired before several liposome-based therapeutics that arecurrently being researched can be translated for widespreadclinical and commercial use

As the costs associated with veterinarymedicine increaseit will be imperative to channel resources into cost-effective

high-efficiency and low-risk drug delivery systems Theaverage veterinary expenditure per household in the US wasabout 366USD per year in 2006 [1] Furthermore it has beenpredicted that the world animal health market will be valuedat 30 billion USD by the year 2020 [50] Therefore liposomesalong with other nanotechnological delivery systems willcontinue to be of importance to veterinary researchers [4 7]The vast potential for liposomes as delivery platforms inanimals has been demonstrated through the studies high-lighted in this review (Table 2) Apart from liposomes nan-otechnological drug delivery vectors also include polymericmicelles ceramic nanoparticles and metallic nanomaterials[4] However most nanoparticles have not been sufficientlyevaluated for in vivo toxicity [51] Liposomes and lipid-based nanoparticles have comparatively few issues withbiodegradability and toxicity [3 4] Furthermore liposomesare highly modifiable and can be studied easily through theirsurface characteristics For instance measuring the zeta-potential or surface charge of cationic liposomes can yieldinformation about their in vivo binding behaviour [34 52]Liposomal vesicles can also easily be sized using photoncorrelation spectroscopy and characterized morphologicallyusing transmission electron microscopy [6 52] Thereforeliposomes serve as highly cost-effective platforms that canbe rapidly formulated and characterized Hence they willcontinue to play an important role in veterinary research inthe future

To conclude it is important to discuss some of thefuture directions of liposome-based research in veterinarymedicine In addition to curative therapies liposomes mayalso be used for dietary supplementation in animals Astudy conducted in postpubertal cows demonstrated that anoral administration of liposome-encapsulated 120572-tocopherolresulted in longer lasting plasma concentrations than otherformulations of this essential vitamin [51] There is a possi-bility in the future of liposomes being used to supplementa broad range of trace minerals and vitamins to preventmorbidity in companion and farm animals Finally lipo-somes are being investigated as platforms for ldquotheranosticsrdquoa term that is a portmanteau of therapy and diagnostics [53]


Incorporating agents that have intrinsic imaging propertiesinto liposomes can create platforms that provide concomitanttherapeutic and diagnostic functions [53] For instance lipo-somes can be engineered to form hybrids with semiconduct-ing nanocrystals called quantum dots (QDs) that have novelmagnetic and imaging properties and also be loaded witha chemotherapeutic agent such as Doxorubicin [53] Thesehave been observed to easily target various organs and havebeen demonstrated to have capacity for in vitro cancer cellkilling at levels similar to freeDoxorubicin [53] Even thoughliposomes were first described in the 1960s they ushered inan age of nanomedicine that has revolutionized the way inwhich veterinary and human researchers perceive the worldof drug delivery Currently it would take a quantum leap innanotechnology for us to be able to construct an intelligentnanobot capable of diagnosing and medicating a patient at amicroscopic level However nanomedicine has taken a stepin that direction with the field of theranostics

References

[1] D J Brayden E J M Oudot and A W Baird ldquoDrug deliverysystems in domestic animal speciesrdquoHandbook of ExperimentalPharmacology vol 199 pp 79ndash112 2010

[2] C Underwood and A W van Eps ldquoNanomedicine and veteri-nary science the reality and the practicalityrdquoVeterinary Journalvol 193 no 1 pp 12ndash23 2012

[3] JM Irache I Esparza CGamazoMAgueros and S EspuelasldquoNanomedicine novel approaches in human and veterinarytherapeuticsrdquo Veterinary Parasitology vol 180 no 1-2 pp 47ndash71 2011

[4] S K Sahoo and V Labhasetwar ldquoNanotech approaches to drugdelivery and imagingrdquo Drug Discovery Today vol 8 no 24 pp1112ndash1120 2003

[5] V P Torchilin ldquoRecent advances with liposomes as pharmaceu-tical carriersrdquo Nature Reviews Drug Discovery vol 4 no 2 pp145ndash160 2005

[6] T Suzuki M Ichihara K Hyodo et al ldquoAccelerated bloodclearance of PEGylated liposomes containing doxorubicin uponrepeated administration to dogsrdquo International Journal of Phar-maceutics vol 436 no 1-2 pp 636ndash643 2012

[7] J S Rose J M Neal and D J Kopacz ldquoExtended-durationanalgesia update on microspheres and liposomesrdquo RegionalAnesthesia and Pain Medicine vol 30 no 3 pp 275ndash285 2005

[8] S C Basu and M Basu Liposome Methods and ProtocolsHumana Press Totowa NJ USA 2002

[9] A N Lukyanov T A Elbayoumi A R Chakilam and V P Tor-chilin ldquoTumor-targeted liposomes doxorubicin-loaded long-circulating liposomes modified with anti-cancer antibodyrdquoJournal of Controlled Release vol 100 no 1 pp 135ndash144 2004

[10] K Kono ldquoThermosensitive polymer-modified liposomesrdquoAdvanced Drug Delivery Reviews vol 53 no 3 pp 307ndash3192001

[11] M Ferrari ldquoCancer nanotechnology opportunities and chal-lengesrdquo Nature Reviews Cancer vol 5 no 3 pp 161ndash171 2005

[12] K H Bae H J Chung and T G Park ldquoNanomaterials for can-cer therapy and imagingrdquoMolecules and Cells vol 31 no 4 pp295ndash302 2011

[13] S Egusquiaguirre M Igartua R Hernandez and J PedrazldquoNanoparticle delivery systems for cancer therapy advances

in clinical and preclinical researchrdquo Clinical and TranslationalOncology vol 14 no 2 pp 83ndash93 2012

[14] D M Vail E G MacEwen I D Kurzman et al ldquoLiposome-encapsulated muramyl tripeptide phosphatidylethanolamineadjuvant immunotherapy for splenic hemangiosarcoma in thedog a randomized multi-institutional clinical trialrdquo ClinicalCancer Research vol 1 no 10 pp 1165ndash1170 1995

[15] I Judson J A Radford M Harris et al ldquoRandomised phaseII trial of pegylated liposomal doxorubicin (DOXILCAELYX)versus doxorubicin in the treatment of advanced or metastaticsoft tissue sarcoma a study by the EORTC Soft Tissue and BoneSarcoma Grouprdquo European Journal of Cancer vol 37 no 7 pp870ndash877 2001

[16] M L Hauck S M La Rue W P Petros et al ldquoPhase I trial ofdoxorubicin-containing low temperature sensitive liposomes inspontaneous canine tumorsrdquo Clinical Cancer Research vol 12no 13 pp 4004ndash4010 2006

[17] M Kleiter A Tichy M Willmann M Pagitz and B Wolfes-berger ldquoConcomitant liposomal doxorubicin and daily pal-liative radiotherapy in advanced feline soft tissue sarcomasrdquoVeterinary Radiology and Ultrasound vol 51 no 3 pp 349ndash3552010

[18] S Hafeman C London R Elmslie and S Dow ldquoEvaluation ofliposomal clodronate for treatment of malignant histiocytosisin dogsrdquoCancer Immunology Immunotherapy vol 59 no 3 pp441ndash452 2010

[19] C R Safinya K Ewert A Anmad et al ldquoCationic liposome-DNA complexes from liquid crystal science to gene deliveryapplicationsrdquo Philosophical Transactions of the Royal Society Avol 364 no 1847 pp 2573ndash2596 2006

[20] G Caracciolo and H Amenitsch ldquoCationic liposomeDNAcomplexes from structure to interactions with cellular mem-branesrdquo European Biophysics vol 41 no 10 pp 815ndash829 2012

[21] P L Felgner T R Gadek M Holm et al ldquoLipofection ahighly efficient lipid-mediated DNA-transfection procedurerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 84 no 21 pp 7413ndash7417 1987

[22] M Rogers and R A Rush ldquoNon-viral gene therapy for neuro-logical diseases with an emphasis on targeted gene deliveryrdquoJournal of Controlled Release vol 157 no 2 pp 183ndash189 2012

[23] L W UrsquoRen B J Biller R E Elmslie D H Thamm and SW Dow ldquoEvaluation of a novel tumor vaccine in dogs withhemangiosarcomardquo Journal of Veterinary InternalMedicine vol21 no 1 pp 113ndash120 2007

[24] D Kamstock A Guth R Elmslie et al ldquoLiposome-DNA com-plexes infused intravenously inhibit tumor angiogenesis andelicit antitumor activity in dogs with soft tissue sarcomardquoCancer Gene Therapy vol 13 no 3 pp 306ndash317 2006

[25] A Schroeder D A Heller M M Winslow et al ldquoTreatingmetastatic cancer with nanotechnologyrdquo Nature Reviews Can-cer vol 12 no 1 pp 39ndash50 2012

[26] Z Wang Y Yu W Dai et al ldquoThe use of a tumor metastasistargeting peptide to deliver doxorubicin-containing liposomesto highly metastatic cancerrdquo Biomaterials vol 33 no 33 pp8451ndash8460 2012

[27] J M Cox and A Pavic ldquoAdvances in enteropathogen control inpoultry productionrdquo Journal of Applied Microbiology vol 108no 3 pp 745ndash755 2010

[28] P Nordly H B Madsen H M Nielsen and C Foged ldquoStatusand future prospects of lipid-based particulate delivery systems


as vaccine adjuvants and their combination with immunostim-ulatorsrdquo Expert Opinion on Drug Delivery vol 6 no 7 pp 657ndash672 2009

[29] T Storni T M Kundig G Senti and P Johansen ldquoImmunityin response to particulate antigen-delivery systemsrdquo AdvancedDrug Delivery Reviews vol 57 no 3 pp 333ndash355 2005

[30] N Csaba M Garcia-Fuentes and M J Alonso ldquoNanoparticlesfor nasal vaccinationrdquo Advanced Drug Delivery Reviews vol 61no 2 pp 140ndash157 2009

[31] K S Korsholm P L Andersen and D Christensen ldquoCationicliposomal vaccine adjuvants in animal challenge modelsoverview and current clinical statusrdquo Expert Review of Vaccinesvol 11 no 5 pp 561ndash577 2013

[32] W Li S Watarai T Iwasaki and H Kodama ldquoSuppression ofSalmonella enterica serovar Enteritidis excretion by intraocularvaccination with fimbriae proteins incorporated in liposomesrdquoDevelopmental and Comparative Immunology vol 28 no 1 pp29ndash38 2004

[33] M E St Louis D L Morse M E Potter et al ldquoThe emergenceof grade A eggs as a major source of Salmonella enteritidis infe-ctions new implications for the control of salmonellosisrdquoJournal of the AmericanMedical Association vol 259 no 14 pp2103ndash2107 1988

[34] K Yaguchi T Ohgitani T Noro T Kaneshige and Y ShimizuldquoVaccination of chickens with liposomal inactivated avianpathogenic Escherichia coli (APEC) vaccine by eye drop orcoarse spray administrationrdquo Avian Diseases Digest vol 4 no2 p e19 2009

[35] K Zhao G Chen X Shi et al ldquoPreparation and efficacy of alive newcastle disease virus vaccine encapsulated in chitosannanoparticlesrdquo PLoS One vol 7 no 12 Article ID e53314 2012

[36] E B Onuigbo V C Okore K C Ofokansi et al ldquoPreliminaryevaluation of the immunoenhancement potential of Newcastledisease vaccine formulated as a cationic liposomerdquo AvianPathology vol 41 no 4 pp 355ndash360 2012

[37] D J Alexander ldquoNewcastle disease in the EuropeanUnion 2000to 2009rdquo Avian Pathology vol 40 no 6 pp 547ndash558 2012

[38] E R Pfefferkorn ldquoInterferon gamma blocks the growth of Tox-oplasma gondii in human fibroblasts by inducing the host cellsto degrade tryptophanrdquo Proceedings of the National Academy ofSciences of the United States of America vol 81 no 3 pp 908ndash912 1984

[39] E Hiszczynska-Sawicka H Li J Boyu Xu et al ldquoInductionof immune responses in sheep by vaccination with liposome-entrapped DNA complexes encoding Toxoplasma gondiiMIC3generdquo Polish Journal of Veterinary Sciences vol 15 no 1 pp 3ndash92012

[40] J R Schmidt L Krugner-Higby T D Heath R Sullivan andL J Smith ldquoEpidural administration of liposome-encapsulatedhydromorphone provides extended analgesia in a rodent modelof stifle arthritisrdquo Journal of the American Association forLaboratory Animal Science vol 50 no 4 pp 507ndash512 2011

[41] M Rathbone and D Brayden ldquoControlled release drug deliveryin farmed animals commercial challenges and academic oppor-tunitiesrdquoCurrent Drug Delivery vol 6 no 4 pp 383ndash390 2009


[43] G J Grant J Lax L Susser M Zakowski T E Weissman andH Turndorf ldquoWound infiltration with liposomal bupivacaineprolongs analgesia in ratsrdquoActaAnaesthesiologica Scandinavicavol 41 no 2 pp 204ndash207 1997

[44] B Richard P Newton L Ott et al ldquoThe safety of EXPAREL(bupivacaine liposome injectable suspension) administered byperipheral nerve block in rabbits and dogsrdquo Journal of DrugDelivery vol 2012 Article ID 962101 10 pages 2012

[45] J R Paul-Murphy L A Krugner-Higby R L Tourdot et alldquoEvaluation of liposome-encapsulated butorphanol tartrate foralleviation of experimentally induced arthritic pain in green-cheeked conures (Pyrrhura molinae)rdquo American Journal ofVeterinary Research vol 70 no 10 pp 1211ndash1219 2009

[46] L Krugner-Higby L Smith B Schmidt et al ldquoExperimen-tal pharmacodynamics and analgesic efficacy of liposome-encapsulated hydromorphone in dogsrdquo Journal of the AmericanAnimal Hospital Association vol 47 no 3 pp 185ndash195 2011

[47] A Livingston ldquoPain and analgesia in domestic animalsrdquoHand-book of Experimental Pharmacology vol 199 pp 159ndash189 2010

[48] D D Frisbie CWMcIlwraith C E Kawcak NMWerpy andG L Pearce ldquoEvaluation of topically administered diclofenacliposomal cream for treatment of horses with experimen-tally induced osteoarthritisrdquo American Journal of VeterinaryResearch vol 70 no 2 pp 210ndash215 2009

[49] J Szebeni C R Alving L Rosivall et al ldquoAnimal modelsof complement-mediated hypersensitivity reactions to lipo-somes and other lipid-based nanoparticlesrdquo Journal of LiposomeResearch vol 17 no 2 pp 107ndash117 2007

[50] P I Menzies ldquoControl of Important Causes of Infectious Abo-rtion in Sheep and Goatsrdquo Veterinary Clinics of North Americavol 27 no 1 pp 81ndash93 2011

[51] J W Card T S Jonaitis S Tafazoli and B A Magnuson ldquoAnappraisal of the published literature on the safety and toxicity offood-related nanomaterialsrdquo Critical Reviews in Toxicology vol41 no 1 pp 22ndash51 2011

[52] B A Yegin and A Lamprecht ldquoLipid nanocapsule size analysisby hydrodynamic chromatography and photon correlationspectroscopyrdquo International Journal of Pharmaceutics vol 320no 1-2 pp 165ndash170 2006

[53] W T Al-Jamal and K Kostarelos ldquoLiposomes from a clinicallyestablished drug delivery system to a nanoparticle platform fortheranostic nanomedicinerdquo Accounts of Chemical Research vol44 no 10 pp 1094ndash1104 2011

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


Table 1 An overview of the morphological characteristics of different types of liposomes [5]

Multilamellar vesicles Consist of several concentric bilayers Range in size from 500 to 5000 nm Ideal for trappinghydrophobic drugs in additional lamellae

Large unilamellar vesicles Consist of one concentric lipid bilayer surrounding a large inner aqueous environment Range in sizefrom 200 to 800 nm Ideal for trapping large amounts of hydrophilic drugs

Small unilamellar vesicles Consist of one concentric bilayer Small size in the range of 100 nm Ideal for long-term circulation

Hydrophilic drug

Hydrophobic drug

PEG

Targeting ligand

Cationic lipid

Phospholipid

Figure 1 A graphical depiction of the versatility of liposomes asdelivery platforms (lowastPEG poly-ethylene glycol)

in veterinary medicine Furthermore nanoparticles devel-oped for and tested in veterinary species may be relevantfor translation to human medicine In fact the pharmacoki-netic and toxicity profiles of nanoparticle formulations areoften tested in canine models [6] Hence liposome-basedtherapeutics that are relevant for veterinary species but alsohave relevance for human nanodrug development will bediscussed Due to the versatile applications of liposomesa review of recent developments in the field is warrantedespecially as it pertains to veterinary applications

2 Liposomes as Delivery Platforms

Liposomes were first described in the 1960rsquos by Alec Bang-ham who reported the ability of phospholipids to formclosed vesicles encircled by lipid bilayers that resemble cellmembranes (Figure 1) [5] The basic structure of liposomesinvolves the hydrophilic head groups of the lipid bilayerdirected towards the aqueous phases whereas the hydropho-bic tail groups are directed towards each other to form the

membrane core [5 7] Generally hydrophobic substances canbe entrapped within the lipid bilayer and hydrophilic sub-stances within the inner aqueous compartment [7] Alteringthe preparation parameters can yield vesicles with differentmorphological characteristics that are shown in Table 1

Liposomes serve as effective delivery platforms due toseveral favourable characteristics (Figure 1) They can encap-sulate both hydrophobic and hydrophilic compounds andcan be used for intracellular drug delivery [7] Moreoverthe vesicle size surface charge and surface properties canbe easily modified using different compounds and prepa-ration parameters [7 8] For example adding polymerssuch as poly(ethylene) glycol (PEG) to the liposomal surface(PEGylation) can create long-circulating liposomes that canevade capture from the reticuloendothelial system (RES)stay in the body longer and demonstrate extended-releaseof the encapsulated drug over time [9] Moreover attachingantibodies and other markers to liposome surfaces canallow for diagnostic imaging and targeted therapy [5 8]Finally liposomes can be designed for triggered release usingexternal stimuli such as pH ultrasound and temperature[5 10] Temperature-sensitive liposomes are designed withthermosensitive polymers that have lower critical solutiontemperatures (LCST) attached to their surface [10] At tem-peratures below their LCST (usually 20∘C) the polymerchains are stable and hydrated but at temperatures higherthan the LCST (at around 39ndash42∘C) they become dehydratedand disrupt the lipid bilayer resulting in an immediate releaseof entrapped contents (Figure 2) [10] The aforementionedcharacteristics of liposomes demonstrate their potential inseveral areas of veterinary medicine In particular liposomescan serve as potent delivery platforms for cancer therapeuticsvaccine and analgesic drugs

21 Liposome-Based Cancer Therapeutics The rationale fornanoparticle based cancer therapeutics has been extensivelyreviewed [11ndash13] Modern cancer therapy involves the use ofseveral antineoplastic agents many of which are chemother-apeutic drugs These drugs are potent at eliminating cancercells in vitro but are observed to have significant barriersto in vivo efficacy [13] These barriers include a lack ofselectivity for cancer cells low bioavailability at tumour siteslarger volumes of distribution and toxicity to normal tissues[12] Nanotechnology-based drug delivery systems such asliposomes can overcome these barriers through a variety ofmechanisms Due to their small size (10ndash100 nm) they areideal for intracellular uptake have high encapsulation capac-ities and can be designed for specific targeting of tumourcells [12 13] Furthermore the intrinsic characteristics oftumour tissue such as leaky microvasculature and highly


ΔT







































References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







ΔT







































References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of




































References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of





























References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






















References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








References
































































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of








































Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






Review Article Recent Developments in Liposome-Based ...downloads.hindawi.com/archive/2013/167521.pdf · review depicts the current signi cance and future directions of liposome-based

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