Quinoline-n-butylcyanoacrylate-based nanoparticles for brain targeting for the diagnosis of Alzheimer's disease

Advanced Review

Quinoline-n-butylcyanoacrylate-based nanoparticles for braintargeting for the diagnosis ofAlzheimer’s diseasePadmakar V. Kulkarni,1∗ Celeste A. Roney,1 Peter P. Antich,1

Frederick J. Bonte,1 Anjanapura V. Raghu2

and Tejraj M. Aminabhavi2∗

A survey of research activity on nanoparticles (NPs) based on polymeric devices thatcould cross the blood–brain barrier (BBB) is given along with the presentation ofour own data on the development of NPs of n-butyl-2-cyanoacrylate (BCA) for braindelivery to aid the early diagnosis of Alzeimer’s disease (AD), a neurodegenerativedisorder of the elderly people, the most prevalent form of dementia. Typical dataare presented on in vivo detection of amyloid peptides (Aβ) (amyloid plaques)that are used as targets for developing the biological markers for the diagnosis ofAD. In order to develop efficient in vivo probes, polymeric n-butyl-2-cyanoacrylate(PBCA) NPs have been prepared and encapsulated with the radio-labeled amyloidaffinity drug 125I-clioquinol (CQ, 5-chloro-7-iodo-8-hydroxyquinoline) to improvethe transport to brain and amyloid plaque retention of 125I-CQ using the NPsof PBCA. The 125I-CQ discriminately binds to the AD post-mortem brain tissuehomogenates versus control. 125I-CQ-PBCA NPs labeled the Aβ plaques from theAD human post-mortem frontal cortical sections on paraffin-fixed slides. Storagephosphor imaging verified preferential uptake by AD brain sections comparedto cortical control sections. The 125I-CQ-PBCA NPs crossed the BBB in wildtype mouse, giving an increased brain uptake measured in terms of % ID/g i.e.,injected dose compared to 125I-CQ. Brain retention of 125I-CQ-PBCA NPs wassignificantly increased in the AD transgenic mice (APP/PS1) and in mice injectedwith aggregated Aβ42 peptide versus age-matched wild type controls. The resultsof this study are verified by in vivo storage phosphor imaging and validated byhistopathological staining of plaques and select metal ions, viz. Fe2+ and Cu2+.The 125I-CQ-PBCA NPs had more efficient brain entry and rapid clearance innormal mice and enhanced the retention in AD mouse brain demonstrating theideal in vivo imaging characteristics. The 125I-CQ-PBCA NPs exhibited specificityfor Aβ plaques both in vitro and in vivo. This combination offered radio-iodinatedCQ-PBCA NPs as the promising delivery vehicle for in vivo single photon emissiontomography (SPECT) (123I) or PET (124I) amyloid imaging agent. The importanceof the topic in relation to brain delivery and other similar type of work publishedin this area are covered to highlight the importance of this research to medicaldisciplines . 2009 John Wiley & Sons, Inc. WIREs Nanomed Nanobiotechnol

∗Correspondence to: [email protected];[email protected] Radiological Sciences, Department of Radiology, TheUniversity of Texas Southwestern Medical Center at Dallas, Dallas,TX 75390, USA

2Center of Excellence in Polymer Science, Karnatak University,Dharwad 580 003, India

DOI: 10.1002/wnan.059

2009 John Wiley & Sons, Inc.

Advanced Review www.wiley.com/wires/nanomed

INTRODUCTION

Nanoparticles (NPs), ranging in size from 10 to1000 nm, were first developed approximately

35 years ago, initially as carriers using biodegrad-able polymers for delivering vaccines and cancerchemotherapic agents.1 Drugs can be adsorbed ontoNP surface, entrapped inside the polymer/lipid or dis-solved within the NPs. In the present day scenario,expectation is rising that nanomedicine and nanotech-nology will bring much greater advances in diagnosingand treating many deadly diseases like cancer. Overthe past decade, progress in developing nanosizedhybrid therapeutics and drug delivery systems hasbeen remarkable.2 The challenge of nanomedicineover other conventional tools of biomedical sci-ences in diagnosing, treating and preventing diseaseshas improved our knowledge in improving humanhealth and hygiene.3 Antibodies and their conjugates,viruses as viral vectors, polymeric micelles, liposomes,polymer-protein conjugates, dendrimers and NPs aresome of the various types of nanomedicines reportedin the literature. It is anticipated that in longer term,nanomedicine will embrace many opportunities aris-ing from stem cell research, tissue engineering as wellas device miniaturization.

Research efforts in recent years in developingnanotechnology-based delivery devices are accelerat-ing at a rapid rate. Nanomedicine, an offshoot ofnanotechnology, has been one of the long-term goalsin the United States National Institutes of Health (USNIH) road map and it anticipates to offer medical ben-efits to the public. In particular, Alzheimer’s disease(AD), a neurodegenerative disorder, principally of theelderly, is one such important issue in nanomedicinearea, especially the early diagnosis of AD. The cogni-tive decline associated with the AD drastically affectsthe social and behavioral skills of the people livingwith this disease. Notwithstanding the social impact,AD also places huge financial burden on patients, fam-ilies and the community as a whole. As is well-known,therapeutic strategies to probe the central nervous sys-tem (CNS) are greatly limited by the restrictive tightjunctions at the endothelial cells of the blood–brainbarrier (BBB).2,4 In order to overcome the imposi-tions of BBB, polymeric NPs have been developed fortargeting the CNS for the treatment of cancer, butstill the drug carrier nanotechnology is yet to be welldeveloped in AD research.4,5 In this task, polymericNPs are the most promising devices for the early diag-nosis of AD because NPs are capable of opening thetight junctions and cross the BBB6,7; thus, they hold agreater promise for high drug loading as well as target-ing the mutant proteins of AD.8,9 The present review

will focus on the discussion of our own experimen-tal data on the development of NPs of cyanoacrylatepolymers for targeting amyloid plaques in the brainalong with a review of other research activities in thisexciting area of research.

Targeted Drug Delivery using NPsThe concept of targeted drug delivery is not new, butdates back to 1906 when Ehrlich first postulated the‘magic bullet concept’. In later years, its clinical use hasmet with challenges mainly on three fronts: (1) findingthe proper target for a particular disease state,(2) finding a drug that effectively treats this diseaseand (3) finding means of carrying the drug in a stableform to specific sites, while avoiding immunogenicand nonspecific interactions that efficiently clear theforeign material from the body. NPs are potentiallyuseful as carriers of active drugs and when coupledwith targeting ligands, may fulfill many attributes ofa ‘magic bullet’. Experimental data on targeted drugdelivery using NPs of n-butyl-2-cyanoacrylates (BCA)to cross the BBB and deliver drug(s) to the brain arediscussed below in relation to the reported data.

Nanoparticulate drug delivery systems havemany advantages, as they can pass through certainbiological barriers like BBB and have the capacity toencapsulate high levels of therapeutic agent. Becauseof their versatilities and preparation methods usedin these systems, surface functionalities can be incor-porated into these NPs. In the present paper, we willdiscuss the recently developed NPs of polymeric(n-butyl-2-cyanoacrylate) (PBCA) for delivering drugs tothe brain. It may however, be noted from a detailedliterature survey that there have been a variety ofother materials that are polymeric in nature and havebeen used to engineer the solid NPs both with andwithout surface functionality. Like many other suchsystems, polylactic acid-polyglycolic acid copolymer(PLGA)-based NPs have been formulated in a varietyof ways to improve the drug pharmacokinetics andbiodistribution to the target sites by either passive oractive targeting.10 Methods of coupling targeting lig-ands to the NPs have also been developed.11 Of these,the most widely used coupling group is polyethyleneglycol (PEG), because this group creates hydrophilicsurface that facilitates the long circulation of NPs.12

Table 1 summarizes some of the typical polymers thatare used to prepare the biodegradable NPs and ourearlier reviews10,12 addressed these issues at length.

The literature cites many examples of NPs ofbiodegradable polybutylcyanoacrylates, as these areproven to pass through the BBB by phagocytosisor endocytosis through endothelial cells. Many


WIREs Nanomedicine and Nanobiotechnology Quinoline-n-butylcyanoacrylate-based nanoparticles for brain targeting

TABLE 1 Typical Polymers used to Prepare BiodegradableNanoparticles for Brain Delivery Research

Type of system Polymer

1. Solid biodegradable polymer Gelatin

Chitosan

PLA, PGA, PLGA

Albumin

PEG-polyester blockcopolymers

Polyanhydrides

Polycaprolactone

2. Vesicular systems Liposome

Niosomes

Polymeric micelles

authors13–17 have shown that therapeutic agents thatnormally do not cross BBB can be transported acrossthis barrier into the brain binding them to PBCANPs coated with polysorbate 80 to facilitate thesuccessful delivery of drugs into the brain. Thishas opened up a new era for the early diagnosisand detection of AD, multiple sclerosis and braintumors in medical research. However, the explosionof interest of biological aspects of AD in recent yearshas led to improvements in the understanding as wellas the treatment of the disease. In this article, someexperimental data on the possibility of targeting drugwith polysorbate 80-coated NPs of PBCA to the brainare presented along with some relevant works fromthe literature supporting the ‘proof of concept’.

The use of NP therefore, effectively protectsthe drug from enzymatic degradation. Differentstrategies have been proposed to allow for NP tocross the BBB. Most of these are on the basis ofthe physical adsorption of a hydrophilic polymer,like the surfactant polysorbate 80 onto the sur-face of polyalkylcyanoacrylate NP.18 Alternatively,the NPs that are able to cross the BBB were alsoprepared using amphiphilic copolymers like theblock copolymer of polyethylene glycol and poly(n-hexadecylcyanoacrylate) (PEG-PHDCA) in whichthe hydrophobic block would form a solid phase,while the hydrophilic part remains on the surface.19

While the mechanism of delivery across the BBBof the NP-coated with surfactants like polysorbate80 seems to be an endocytotic uptake via the lowdensity lipoprotein (LDL) receptor by the endothelialcells, mediated by the adsorpion on the NP ofapolipoprotein B and/or E from the blood.20,21 ThePEGylated PHDCA NPs have also shown to penetrateinto the brain probably due to their long-circulating

characteristics.19 If the PBCA NPs are not coveredwith the surfactants, then they get retained in theblood vessels.18 Thus, their ability to cross the BBB islinked only to the chemico-physical characteristics ofthe surface of NPs.

PBCA NPs have been reported in the literaturemostly to achieve successful delivery of various typesof drugs to the brain with the help of surface-coatedsurfactant. Polysorbate 80 was particularly found tobe the most efficient surfactant among many surfac-tants tested.22,23 However, the favored transport ofNPs coated with polysorbate 80 follows the receptor-mediated endocytosis by the brain endothelial cells;polysorbate 80 preferentially absorbs apolipoproteinE (Apo-E) in the plasma and then NPs coated withApo-E are recognized as LDL that are internalizedby the LDL uptake system.24,25 Thus, polysorbate80-coated PBCA NPs loaded with anti-cancer doxoru-bicin drug have been investigated intensely for testingthe delivery efficiency, therapeutic efficacy as well asbiodistribution by in vivo animal studies.26,27 In mostof these studies, PBCA NPs were synthesized by theanionic emulsion polymerization. Biodistribution ofPBCA NPs was investigated after the i.v. injectionof three different 14C radio-labeled NP preparationsinto glioblastoma 101/8-bearing rats.28

BBBBBB is the homeostatic defense mechanism of thebrain against pathogens and toxins. Being highlycomplex and regulated, BBB functions as an effectivebarrier that selectively allows the passage of desiredmolecules into the brain parenchyama. However,BBB restricts the entry of solute molecules into thebrain by transcellular route because of increasedelectrical resistance between endothelial cells at thetight junctions. Transport mechanisms at the BBB canthus be manipulated for the cerebral drug targeting.However, it is greatly anticipated that appropriatesurface modification of NPs can deliver drugs ofinterest beyond BBB for diagnostic applications ofneurological disorders. The biology of BBB has beendiscussed earlier in our published review.4 It is nowevident that BBB regulates the passage of moleculesbetween the bloodstream and the brain. However,overcoming the difficulty of delivery drugs to specificareas of the brain is a major challenge. The BBB exertsa neuroprotective function as it hinders the deliveryof diagnostic and therapeutic agents to the brain.

In this paper, we provide an overview of theway in which peptides and nanotechnology are beingexploited in tandem to address the problem of braintargeting. Peptides can be used as specialized coatings



able to transport the NPs with specific targeting, asNPs can also carry the peptide drug. Furthermore,peptides can be used in less conventional approachessuch as all-peptide NPs. However, the combined useof peptides and nanotechnology seems to offer atremendous hope in the treatment of brain disorders.29

Thus, the BBB, together with the blood-cerebrospinal-fluid barrier, protects and regulates the homeostasisof the brain. However, these barriers also limitthe transport of small-molecules and, particularly,biopharmaceutical drugs such as proteins, genes andinterference RNA (SiRNA) to the brain, therebylimiting the treatment of many brain diseases. Asa result, various drug delivery and targeting strategiesare currently being developed to enhance the transportand distribution of drugs into the brain. It maybe noted that we will not attempt to discuss thebiology and physiology of BBB for drug transport tothe brain even though many studies have addressedthe possibilities of delivering large-molecule drugs,particularly genes, by receptor-mediated nonviral drugdelivery to the (human) brain.30

Clioquinol (CQ) as the Metal-ComplexingAgentMarkedly elevated concentrations of zinc, copper andiron in amyloid deposits of the AD brain are well doc-umented in the literature.31,32 Cherney et al.25 haveemployed the antibiotic and Cu/Zn-selective metalchelator, CQ, to inhibit Aβ accumulations in the ADAPP2576 transgenic (Tg) mice. Oral treatments withCQ reduced the Aβ deposition by ≈49%, which pro-duced no neurotoxicity in a blind study. Furthermore,Ahi et al.33 speculated that CQ’s action on peptidemay facilitate H2O2inhibition. They also validated theselectivity of CQ, as systemic metal depletion was notfound and CQ did not deplete the brain tissue of met-als, but rather binds to the Aβ-metal complex itself.

In vivo experiments with non-transgenic animalstreated with CQ confirmed a significant decrease incerebral concentrations of Cu, Zn and Fe metal ions.34

Cherney et al.25 studied the of APP2576 Tg mice, theinhibition of Aβ deposition by CQ caused significantincrease in the cerebral concentrations of Cu and Zn.This is because APP transgene expression reduced theCu and Zn levels in vivo, while the Aβ concentrationsteadily increased.27 This may be attributed to the factthat metal ions that are effluxed by Aβ or APP/Aβ

might have prevented the re-uptake of the ions.28

However, CQ might have prevented the uptake ofmetals by the protein. This action afforded the metal-lic access to the peripheral brain tissue and hence,metal ion concentration was increased. As AD is a

syndrome of metal dyshomeostasis, CQ may be ableto restore the metal metabolism to its normal state.

CQ is a small lipophilic neutral molecule;therefore, it is expected to cross the BBB. However, itwas not exported into the brain efficiently, presumablybecause of its high degree of nonspecific bindingto the plasma proteins, particularly albumin. Opazoet al.35 reported that brain uptake of 125I-CQ was<1% percent injected dose per gram of the tissue(ID/g) in control and transgenic mice within 5 minof administration of the tracer. Their pilot data fromthe human subjects with AD and controls indicatedthat 123I labeled CQ uptake in AD lesions was toolow to permit single photon emission tomographic(SPECT) imaging. They further postulated thatstrategies to increase tracer delivery or modifying theformulation might enable the imaging of plaques inthe AD patients. Therefore, we have investigated thepossibility of an appropriate NP-based drug carriersystem to improve the brain transport of 125I-CQ.Polymeric butylcyanoacrylate NPs were chosen to bethe drug carrier in the present study. We found that125I-CQ-BCA NPs act as the effective targeted drugcarrier, showing an affinity for the amyloid plaques.Our studies indicated that 125I-CQ-PBCA NPs are themost promising candidates for in vivo brain imagingof amyloid plaques, while other studies have proventhe successful utilization of PBCA NPs for other classesof drugs.13–17 It may be noted that the use of CQ wasrecently stopped because of some toxicity concerns(optic neuropathy) and also because it was attributedto vitamin B12 deficiency. However, the amount ofmaterial administered for diagnostic purposes is verysmall, (usually nm to µm range). Thus, it is necessaryto test the toxicity of NPs along with the activeingredient before pursuing further studies.

Agdeppa et al.36 present a comprehensive reviewon the in vitro and in vivo detection of neurofibrillarytangles (NFTs) and β-amyloid senile plaques (SPs),neuropathological lesions found in the brains ofthe AD patients, using 2-(1{6-[(2-[18F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile([18F]FDDNP) and its analogs. [18F]FDDNP and itsanalogs have shown excellent ability to bind to NFTsand SPs in vitro as shown through the binding assaystudies by a confocal fluorescence microscopy withstained AD brain tissue. The digital autoradiographywith [18F]FDDNP, [18F]FDDNP-PET molecularimaging detected these pathologies in living subjects.Thus, the discovery of a new binding site to Aβ(1-40)fibrils as a result of FDDNP binding opens up a noveltherapeutic opportunity for the early diagnosis ofAD. FDDNP thus, shares a previously unrecognizedcommon binding site on Aβ(1-40) fibrils and SPs



with non-steroidal anti-inflammatory drugs (NSAIDs)(e.g., naproxen and ibuprofen). Naproxen, ibuprofenand even FDDNP significantly inhibit the aggregationof Aβ(1-40) peptide in the micromolar range. Thisnew binding site on Aβ(1-40) fibrils also offers amolecular template for designing anti-aggregationof drugs without showing any secondary effects ofNSAIDs. Therefore, it is anticipated that a new visionfor the prevention and early diagnosis of AD wouldbe rapidly available.

The 6-iodo-2-(4′-dimethylamino-)phenyl-imid-azo[1,2-a] pyridine [123I-IMPY] is also found to be anovel radiopharmaceutical that selectively binds to theAD amyloid plaques. As a first step toward validatingthis radiopharmaceutical as an imaging biomarkerfor AD, Newberg et al.37 measured the whole-bodybiokinetics and radiation dosimetry of 123I-IMPY inAD patients and cognitively normal control subjects.The pharmacologic safety profile of the compoundwas simultaneously assessed. The sample includednine subjects ranging in age from 44 to 80 years.Whole-body images were obtained for each subjectfor up to 48 h after the intravenous administrationof 185 MBq (5 mCi) of 123I-IMPY. The fraction ofadministered activity in 12 regions of interest wasquantified from the attenuation-corrected geometricmean counts in conjugate views. Multiexponentialfunctions were iteratively fit to each time-activitycurve using a nonlinear, least-squares regression algo-rithm. These curves were numerically integrated toyield the cumulated activity values for source organs.The radiation doses were then estimated with themedical internal radiation dose (MIRD) technique.

The radiotracer had no pharmacologic effects(produced no changes in heart rate, blood pressure,or laboratory results) on any of the subjects.Radiation dosimetry estimates indicated that the dose-limiting organ was the gallbladder, which receivedan average of 0.135 mGy/MBq (range, 0.075-0.198mGy/MBq). The effective dose equivalent and effectivedose for 123I-IMPY were 0.042 ± 0.003 mSv/MBqand 0.035 ± 0.001 mSv/MBq, respectively. Themean effective dose for 123I-IMPY was similarto that for 111In-diethylenetriaminepentaacetic acid(0.035 mGy/MBq), less than half that for 111In-pentetreotide (0.81 mGy/MBq) and approximatelytwice that for 123I-IMP (0.018 mGy/MBq). Nosignificant differences were found between men andwomen or between AD patients and control subjects.Therefore, the 123I-IMPY may be a safe radiotracerwith appropriate biokinetics for imaging amyloidplaques in AD patients. Thus, overall, the technologybase is available for BBB targeting of drugs throughother means also. In the present research, we have

studied the in vivo storage phosphor imaging andvalidated by histopathological staining of plaquesand metal ions like Fe2+ and Cu2+. The 125I-CQ-PBCA NPs had more efficient brain entry andrapid clearance, in normal mice and enhanced theretention in AD mouse brain demonstrating the idealin vivo imaging characteristics and also exhibiting thespecificity for Aβ plaques both in vitro and in vivo.This combination offered the use of radio-iodinatedCQ-PBCA NPs as the promising delivery vehicle forin vivo SPECT (123I) or PET (124I) amyloid imagingagent. These reports suggested the importance of thetopic in relation to brain delivery. However, othersimilar types of works published in this area are oflesser importance to highlight the importance of thisresearch to medical disciplines.

METHODS

Radio-iodination of CQCQ was radio-labeled with 125I (Perkin Elmer) by theChloramine-T (CT) method of radio-iodination. Allexperiments were carried out under radiochemistryhood, behind lead shielding; reagents were of analyt-ical grade, purchased from Sigma Chemical Co. (St.Louis, MO). Briefly, 5-chloro-8-hydroxy quinoline(precursor of CQ, 1µg in 10 µL ethanol) was addedto a 16 × 100 mm reaction vessel containing 50µLof 0.5M phosphate buffer (pH 7.4) and 1–2 mCiof 125I (NaI). CT (20 µg in 20 µL water) was usedas the oxidizing agent to release I2 from NaI. Thereaction mixture was briefly vortexed (2 min) beforebeing stopped by the addition of sodium metabisulfite(50 µg in 50 µL water added as the reducing agent).Purification of 125I-CQ was performed by solventextraction in dichloromethane (DCM). Organiclayer (e.g., DCM) was evaporated under N2gas and125I-CQ was dissolved in dimethylsulfoxide (DMSO,1 mL). Purified 125I-CQ was analyzed by radio thinlayer chromatography (RTLC) and had radiochemicalpurity >95%. The product was diluted in phosphatebuffered saline (PBS) (pH 7.4) to appropriate concen-tration before using for in vitro or in vivo studies.

Preparation of PBCA NPsNPs of PBCA were prepared by different polymer-ization techniques for in vivo biodistribution studiesin order to find an appropriate candidate for in vivoimaging of amyloid beta plaques. Briefly, CQ wasradio-iodinated (as discussed above) and incorporatedwithin PBCA NPs for in vivo labeling of amyloidplaques in AD transgenic mice. NPs were polymerizedas per the modified procedure of Kreuter et al.31



Briefly, acidic polymerization medium containing dex-tran 70,000 and polysorbate 80 (Tween-80) was used(both at a concentration of 1% each in 0.1 N HCl)(Sigma, USA); 125I-CQ was (0.1–0.5 mCi) addedto the solution just prior to the addition of butyl-cyanoacrylate (BCA) monomer. Butylcyanoacrylate1% (Sichelwerke, Hannover, Germany) was addedunder constant magnetic stirring at 400 rpm speed.After 3h of polymerization, the NP suspension wasneutralized with 0.1 N NaOH to complete the poly-merization. This solution was filtered through 0.2 µmfilter and purified by ultracentrifugation (Beckman,USA; 45K rpm, 1h). The pellet was washed and redis-persed in water, which contained 1% Tween-80.

PBCA NPs formed by the above procedure weresurface-coated with 1% Tween-80 by stirring for30 min in PBS just before in vivo administration.A 1 mg solution of NPs was administered byintravenous injection into mice. These details wereworked out as per the protocols given elsewhere.17,31

The particle size was measured by Zeta-sizer 3000HS (Malvern, UK). The size of the particles measuredwas found to be around 50 (±5) nm. Encapsulationefficiencies varied from 50 to 60%. Radiochemicalpurity of the radiolabeled NPs was >95% as measuredby the RTLC. There was no change in the particle sizeprofile (measured by dynamic light scattering) whenparticles were prepared by loading with equivalentquantity of unlabeled CQ or no drug. Non-radioactiveNPs were analyzed by scanning electron microscopy(SEM) or transmission electron microscopy (TEM).These analyses showed the particles to be uniformlyspherical in nature.

In Vitro Labeling of Amyloid PlaquesIn vitro assays were performed by incubating humanpost-mortem (1 mg) cortical frontal AD and controlbrain tissue [15 min incubation time, 800 µL buffersolution of 0.1% FBS (fetal bovine serum in PBS)]in the presence of 125I-CQ (1–2 µCi in 100 µL PBS).Brain homogenates were microcentrifuged (13K rpm,

15 min) and the % binding was calculated. Exper-imental results provide an evidence of preferentialbinding by 125I-CQ to AD brain tissue compared tocortical control brain tissue. Procedures followed inthis step are the same as given before.17,31

In Vivo Biodistribution of 125I-CQ BCANPs and 125ICQRadio-labeled NPs or the free drug (125ICQ) (1–5µCi) was administered intravenously by lateral tailvein injection into normal BALB/C male mice (25 g).Group of animals (n = 5) were sacrificed at varioustime intervals (2 min to 4 h post administration of thetracers). Various tissues were taken out, washed withsaline, wiped dry, weighed and counted in a gammawell counter (70% efficiency). Blood was taken out bythe cardiac puncture. An aliquot of suitably dilutedtracer was counted along with the samples and % ID/gwas calculated. These data are presented in Tables 2and 3.

Aggregation of Aβ42 PeptideAmyloid protein (1-42), Aβ42 (0.5 mg) was purchasedfrom Bachem California (Torrance, CA) and dissolvedin 1.15 mL of PBS (pH 7.4) to a final concentrationof 435 µg/mL (100 µM) by magnetically stirring ina closed vessel on a magnetic stirrer at 1200 rpmfor 7 days at room temperature.32 After 7 days, theaggregated peptide suspension was visibly cloudy. Theaggregated Aβ42 was stored in 100 µL aliquots at−20◦C until further use.

Intrahippocampal Sterotaxic Injection ofAggregated Aβ42The use of animals was in compliance with the regu-lations of the Animal Resources Center (ARC) of UTSouthwestern Medical Center, Dallas and approvedby the Institutional Review Board (IRB). Wild typeBALB/C mice (Charles River Laboratories) were

TABLE 2 In vivo Bio-distribution of 125I-CQ by IV Administration in Wild Type Mice (n = 5)(Table Represents Organ Uptakes in % ID/g ± SD)

Time (min) Brain Blood Spleen Liver Brain : blood ratio

2 1 ± 0.4 9.2 ± 2.8 2.6 ± 0.1 12.5 ± 0.8 0.1 ± 0.1

5 1 ± 0.3 5.7 ± 1.3 N/A 15.5 ± 5.6 0.2 ± 0.01

15 0.3 ± 0.1 2.9 ± 0.7 1.2 ± 0.1 3.8 ± 0.7 0.1 ± 0.1

30 0.3 ± 0.1 2.3 ± 0.4 1.3 ± 1 5.2 ± 2.2 0.1 ± 0.1

60 0.1 ± 0.1 1.3 ± 0.7 0.5 ± 0.3 3.6 ± 2.3 0.3 ± 0.5

120 0.03 ± 0.03 0.1 ± 0.1 0.6 ± 0.4 1.3 ± 0.5 0.2 ± 0.1

240 0.04 ± 0.02 0.6 ± 0.2 0.4 ± 0.3 0.9 ± 0.2 0.1 ± 0.1



TABLE 3 In vivo Bio-distribution of 125I-CQ-BCA-NPs by IV Administration in Wild Type Mice(n = 5)

Time (min) Brain Blood Spleen Liver Brain : blood ratio

2 2.3 ± 0.9 12 ± 2.5 1.6 ± 0.4 15 ± 2.1 0.2 ± 0.1

5 1.5 ± 0.9 7.9 ± 1.6 1.8 ± 0.5 11.4 ± 1.5 0.2 ± 0.1

15 0.5 ± 0.2 4.9 ± 1.2 1.4 ± 0.5 8.1 ± 2.1 0.1 ± 0.1

30 0.3 ± 0.1 3.4 ± 1.5 0.9 ± 0.7 6.6 ± 2.4 0.1 ± 0.0

60 0.2 ± 0.1 2.1 ± 1.1 0.5 ± 0.3 3.9 ± 1.6 0.1 ± 0.0

120 0.03 ± 0.0 0.7 ± 0.2 0.2 ± 0.03 2.3 ± 0.4 0.1 ± 0.0

240 0.02 ± 0.0 0.4 ± 0.0 0.1 ± 0.1 1.4 ± 0.1 0.01 ± 0.0

NP Polymerized in the Presence of 1% Dextran 70,000 and 1% Tween-80; Table Represents OrganUptakes in % ID/g ± SD

injected by direct stereotaxis (Model 900 Small Ani-mal Stereotaxic Instrument, David Kopf Instruments,Tujunga, CA) with the aggregated Aβ42 peptide at theconcentration of 1 µg/1 µL under the constant flowrate of 60 s. The mice received unilateral injections ofeither saline or Aβ peptide; the injection location cor-responded to the mouse brain hippocampus structureCA1 at coordinates −1.5, −1.0 and −1.8 relative toBregma.33,34 The animals were allowed to recover for7 days during and after which time the cognitive testsfor the behavior (Y-maze) were performed.

Y-Maze Testing of Cognitive BehavioralSkillsShort term memory, also called working spatialmemory, was assessed in the AD transgenic mice(APP/PS1 and APP/PS1/Tau) and in the mice periph-erally injected with the aggregated Aβ42 peptideby statistically recording the animal’s behavior ina Y maze.38 Cognitive behavioral skills were deter-mined on-going in the AD transgenic mice and theage-matched controls. The age range of the transgenicmice was 5–21 months. Cognitive behavioral skillswere determined at 0, 3 and 7 days post injection ofthe Aβ42 peptide. The age of the peripherally injectedmice was 8 weeks.

The Y maze was constructed of black paintedwood and the length of each arm was 40 cm, whilethe height was 13 cm. The arms (labeled A, B andC) were placed at equal angles to form the Y. Themice were consistently placed at the end of arm A; thespontaneous activities of each mouse were individuallyvisually recorded during 8 min timed sessions, wherean alternation trial was defined as the consecutiveentry into each of the arms. For example, ABC,CBA, BCA, etc., were defined as alternation trials,whereas CCB, BBA, AAC, etc., were not consideredas alternation trials. Arm entry was defined when both

hind feet crossed the boundary of an arm. Entry atany time into the triangle defining the intersectionof the three arms was not considered for statisticalcalculation of the results. Likewise, the initial entryinto arm A was not considered for statisticalcalculation of the results. Thus, the maximum numberof entries was the total number of arm entries minustwo. The % alternation was then calculated as

%Alternation = Total alternation trials/

(Maximum arm entries − 2) × 100%(1)

In Vivo Imaging of Plaques by NanoparticleAdministrationThe 125I-CQ-BCA NPs (1–3 mg, containing 1–5 M,counts per minute (CPM) activity) were administeredto AD mouse models by lateral tail vein injection.The Aβ42 mice received 125I-CQ-BCA NPs at8 days post-injection of the peptide. In vivo storagephosphor autoradiography (Perkin Elmer CycloneStorage Phosphor Imaging System; OptiquantImaging Software) was used to determine the relativequalitative differences in the brain uptakes of 125I-CQby the AD mouse models and wild-type mice, usingthe Optiquant Imaging Software. Briefly, eachmouse was anesthetized I.P. with 100–150 µL ofKetamine HCl (Sigma Aldrich, USA). Animals wereimaged at various time intervals (viz. 5, 15, 30, 60and 90 min post administration of the tracer) andeach imaging session lasted for 5–7 min. Animalswere kept still during the imaging sessions; additionalanesthesia was used if necessary. Post-injection of theradio-labeled NPs mouse was placed on a phosphorscreen with a lead sheet between the film and theanimal’s body in such a way that the only exposedbody part to the screen was the head. In this way,background radiation from the whole body was



minimized and activity source projected onto thefilm was from animal’s head region only. Regions ofinterest were drawn in the brain space to obtain theinformation in semi-quantitative units (dynamic lightunits, DLU) per volume space (DLU/mm2).

At the conclusion of the imaging experiments,the mice were sacrificed by cardiac perfusion throughthe left ventricle with 4% paraformaldehyde. Brainswere harvested and stored in formalin, before beingembedded in wax and cut at a 5 µm slice thickness. Theslices were stained with Congo red (amyloid plaques),Prussian blue (Fe2+) and Rubeanic acid (Cu2+).

AD Transgenic MiceMice with a double mutation (APP/PS1) for AD werepurchased form Jackson Laboratories USA [strainname B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J)]. Thisparticular mouse model corresponds to a form of earlyonset disease and expresses a mutant human presenilin1 and a chimeric mouse/human amyloid precursorprotein (APPSwe). The expression of both transgeneswas directed by the mouse prion protein promoter.The APPswePS1 strain was developed on a B6C3HF2background. The chimeric APP was modified toencode the Swedish mutations K595N/M596L inorder to elevate the amount of Aβ produced from thetransgene, by favoring processing through the beta-secretase pathway. Mice with the double mutation(APP/PS1) were generously donated by Dr DavidRussell (UT Southwestern).

RESULTS AND DISCUSSION

In Vitro Binding of 125I-CQ and 125I-CQBCA NPs to Brain TissueExperimental results provide an evidence of preferen-tial binding by 125I-CQ to the AD brain tissue (1000µg, 18.5% binding) as compared to cortical controlbrain tissue (1000 µg, 13% binding). Thus, it wasshown that an amyloid-affinity drug could be suc-cessfully radio-labeled; the radio-ligand discriminatedbetween AD brain tissue and control brain tissue.Figure 1 shows the labeling of post-mortem humanAD and control frontal cortex brain tissue sections(Slides). NPs loaded tracer showed preferentialuptake in AD brain section by binding to amyloidplaques.

In Vivo Biodistribution of 125I-CQ BCA NPsBCA monomer was polymerized with small particu-late diameter (45 nm) and with a uniform size distri-bution. In vivo bio-distribution experiments showed

AD Frontal cortex Control frontal cortex

FIGURE 1 | In vitro phosphor screen images of cortical brain (fixed)slides from postmortem AD and control subjects. The slides weredeparaffinated first by immersion in xylene and ethanol, and then theywere incubated with 125I-CQ BCA Nanoparticles (2 × 106 CPM in0.5 mL PBS buffer per slide); incubation time: 30 min; phosphor screenfilm exposure time: 7 min. Notice the enhanced uptake of the tracer inAD brain section compared to control brain section.

that the free 125I-CQ had a rapid brain uptake as wellas rapid blood clearance in normal mice (Table 2).Furthermore, 125I-CQ cleared the brain quickly; the% ID/g for the brain at 2 min was 0.99 ± 0.40%,while at 4 h, it was 0.04 ± 0.02%. Table 3 shows thatwhen 125I-CQ was encapsulated within the BCA NPs,the brain uptake was enhanced. At two minutes, thewild type mice exhibited 2.31 ± 0.89% uptake of 125I-CQ BCA NPs in the brain (Table 3). Brain and bloodclearances of the 125I-CQ BCA NPs were rapid; the %ID/g (brain) at 4 h was 0.02%. From Tables 2 and 3,it is observed that 125I-CQ BCA NPs have an increasedbrain uptake versus 125I-CQ in the wild type mice.

Y Maze Testing of Cognitive SkillsWild type mice were intracranially injected with theaggregated Aβ42 peptide and placed in a Y mazeto study the cognitive effects of the peptide. Theseartificially produced mice did not show any signs ofcognitive decline when compared to normal mice thatwere injected with saline. The injected Aβ42 micewere typically challenged through the maze before theinjection of the peptide, and again at 3 and 7 dayspost surgery. They were sacrificed after seven daysand thus, the life span of the disease may have beentoo short to properly assess the challenge of the mazeon the cognitive behavior viz., the working spatialmemory (short term memory).

The cognitive behavioral skills of the ADtransgenic mice (APP/PS1) were also challenged usingthe Y maze. In the 5 months AD transgenic mouse, the% testing range was 54.52–61.44. In the 18 monthswild type mouse, the % testing range was 49.66–65.66. Thus, it was found that the 5 months old



Abeta-42 aggregates injected animal

Control

FIGURE 2 | Storage phosphor images of heads of mice, injected with125I-CQ BCA NPs. 12 min post injection of (∼3 mg of NPs containing7 × 106 CPM) 125I-CQ-BCA NPs: a. mouse intracranially administeredwith 5 µg, A-beta (Aβ42) peptide aggregates 1 week prior toadministration of the tracer. b. control mouse had saline administration.

AD transgenic mice had the equivalent short termmemory of 18 months old wild type mice.

In Vivo Imaging of the Amyloid Plaques inAD Mouse ModelsPBCA NPs were successfully loaded with the radio-labeled quinoline derivative 125I-CQ and deliveredto the mice by intravenous administration. Storagephosphor imaging qualitatively showed that NPstransported the drug across the BBB. Further, the125I-CQ labeled the amyloid deposits. Figure 2 showsthe preferential retention of 125I-CQ BCA NPs inan animal with intracranial administration of Aβ-42peptide aggregates compared to the control animalinjected with saline. Figure 3 displays the in vivostorage phosphor screen images of the brain uptakesof 125I-CQ BCA NPs and 125I-CQ in transgenic mice

AD transgenic mouse injected with 7.56 x 105 CPM 125ICQ BCA NPs

AD transgenic mouse injected with 8.06 x 105 CPM 125ICQ

Mice aged 7 months; imaged 15 min post injection

AD transgenic mice injected with 125ICQ BCA NPs and 125ICQ

FIGURE 3 | Storage phosphor images of heads of transgenic miceinjected with 125CQ BCA NPs and 125I-CQ (∼1M CPM activity in 0.5 mgNPs), 15 min post administration of the tracers. Notice the enhancedactivity retention in animal injected with the nanoparticles compared toanimal injected with the free drug.

125ICQ 125ICQ BCA NPs

Brain images were obtained 90 minutes post injection

12 months old AD transgenic mice injected with 125ICQ or 125ICQ BCA NPs

FIGURE 4 | Storage phosphor images of heads of transgenic miceinjected with 125CQ BCA NPs and 125I-CQ (∼1M CPM activity in 0.5 mgNPs), 90 min post administration of the tracers. Notice the enhancedactivity retention in animal injected with the nanoparticles compared toanimal injected with the free drug.

(7 months old), at 15 min post administration of theNP-encapsulated drug and the free drug. The ADtransgenic mouse has a greater brain uptake withthe use of NPs as compared to free 125I-CQ; thus,encapsulation of 125I-CQ by NPs would enhance theBBB crossing of the drug. Furthermore, encapsulationof 125I-CQ by NPs enhances the retention of the drugin transgenic mice.

Figure 4 displays the in vivo phosphor screenimages of the brain uptakes of 125I-CQ BCA NPsand 125I-CQ in transgenic mice (7 months old) at90 min post administration of the NP-encapsulateddrug and the free drug. At 90 min post injection, theAD transgenic mouse has a greater brain retention ofthe tracer encapsulated in the NPs as compared to thefree 125I-CQ. Transgenic mouse has a higher brainretention of the NP delivered drug as compared to thefree drug indicating that NPs not only enhanced thebrain delivery, but also had longer retention possiblybecause of the binding to amyloid plaques. Figure 5

Control mouse AD mouse

Brain images of control and AD transgenic mice injected with 125ICQ BCA NPs 1 hour post injection

FIGURE 5 | Storage phosphor images of heads of transgenic miceinjected with 125CQ BCA NPs (∼2 M CPM activity in 1 mg NPs), 60 minpost administration of the tracer. Notice the enhanced activity retentionin AD transgenic mouse compared to control animal.



Congo red staining of amyloid plaques: 15 month old AD transgenic mouse (10 µm slice)

FIGURE 6 | This figure shows the Congo red histological staining ofbrain slice from 15 month old AD transgenic mouse. Amyloidaggregates are identified by the Congo red stain.

represents the in vivo phosphor screen images of thebrain uptakes of 125I-CQ BCA NPs in the 12 monthsold wild type mouse and in the 15 months old ADtransgenic mouse. At 1 h post injection, the ADtransgenic mouse has increased the brain retentionof NPs, presumably because of binding of the tracerto amyloid plaques. The histological staining of brainslices taken from the AD mouse models verified thepresence of amyloid plaques by Congo red staining asshown in the Figure 6. Presence of Fe3+ in amyloidregion was confirmed by staining with Prussian blueas seen in the Figure 7.

In the present study, the PBCA NPs werespecifically designed for carrying amyloid affinitydrug 125I-CQ across the BBB in mouse models of AD.In vivo bio-distribution of the 125I-CQ encapsulatedin polybutylcyanoacrylate NPs in the wild-type mouseshowed that they crossed the BBB with a greaterefficiency than the 125I-CQ control. The BCA NPwas selected as the prototype drug carrier because itwas polymerized with the highest reproducibility; itcrossed the BBB and showed rapid uptake as well asclearance from the normal brain. These parametersare important in validating the PBCA NPs that aresurfactant-coated with Tween-80 as a drug carrier tocross the BBB.

The prototype PBCA NP was fully characterizedfor its physicochemical properties and stabilizer effect.For example, smaller sized particles resulted when themonomer was polymerized at a lower pH (pH 1) in thepresence of the stabilizer, dextran 70,000 (as opposedto hydrophilic PEG), and definitely in the presence ofa surfactant (e.g., Tween-80). Loading of NPs with

Fe3+ staining of hippocampal amyloid plaques: 15 month old AD transgenic mouse (5 µm slice)

FIGURE 7 | Prussian blue staining of hippocampus of AD transgenicmouse. Prussian blue shows the presence of Fe3+ in amyloid plaques.Amyloid plaques were identified by staining with Congo red.

amyloid-affinity drugs such as thioflavins (S- or T-)or Congo red did not significantly affect the size ofthe PBCA NPs. Therefore, these NPs maintained theirstability upon drug loading in vitro.

Amyloid-affinity drug CQ was radio-iodinatedwith 125I. The 125I-CQ was used for in vitro assay ofhuman post-mortem frontal cortex to test the affin-ity of the radio-labeled tracer for amyloid plaques.Autoradiography validated the preferential labelingof AD tissue by 125I-CQ (compared to control braintissue). Then, 125I-CQ was successfully encapsulatedwithin PBCA NPs. The 125I-CQ PBCA NPs preferen-tially labeled the human post-mortem AD brain tissuecompared to the control frontal cortex tissue. Theseexperiments were validated by storage phosphorscreen imaging technique. Therefore, PBCA NPs actedas the drug carriers of 125I-CQ, targeted towards amy-loid plaques, presumably binding to the transitionalmetals-peptide aggregates in the amyloid plaques.

The results of this study are quite significant forthe future prospects of in vivo imaging in AD. Thepresent in vitro experiments provided a platform forsmall animal in vivo imaging using 125I-CQ PBCANPs as targeted drug carriers. The imaging of amyloidplaques was demonstrated in two mouse models using125I-CQ BCA NPs as an in vivo imaging vector. Themouse models were AD transgenic mice (APP/PS1)and mice artificially injected with the Aβ peptideaggregates by direct intrahippocampal injection. TheAD mouse models showed consistent and reproducibleresults of increased brain uptake/retention of 125I-CQ PBCA NPs versus free 125I-CQ. Imaging resultswere confirmed by neurohistological staining of the



harvested brain slices (AD and control). The AD brainslices were found to be positive for metal ions (e.g.,Fe2+ and Cu2+), consistent with 125I-CQ interactionwith the amyloid plaques via the metal association.Thus, it is evident that the 125I-CQ PBCA NPsdemonstrate an effective method for in vivo and exvivo detection of amyloid plaques in mouse modelsof AD.

In the past thirty years, nerve growth factor(NGF) has received much attention for its potentialrole as a therapeutic agent for AD due to itsneurotropic activities on basal forebrain cholinergicneurons. This attention has been renewed by recentfindings that provide new links between defects inNGF signaling, transport or processing to the activa-tion of the amyloidogenic route and, more generally,to AD neurodegeneration. Thus, the concept of ther-apeutic administration of human recombinant NGFin AD patients has a strong rationale, being furthervalidated by recent and ongoing clinical trials withthe gene-therapy approach. However, the widespreadclinical application of gene or cell-therapy strategiesfor the delivery of NGF to AD patients seemsunpractical, and it would be more advantageous tohave non-invasive methods, that should also limitthe adverse effects of NGF in activating nociceptiveresponses. A recent review discussed the results frompreclinical and clinical studies underlying the rationaleof NGF as the potential therapeutic agent for AD.39

Alternative strategies to reach adequate concentra-tions of NGF in relevant brain areas while preventingthe onset of adverse effects were also discussed.

In an effort to design a smart nanovehicle (SNV)capable of permeating through the BBB to target cere-brovascular amyloid in both AD and cerebrovascularamyloid angiopathy (CAA), chitosan-based particlesprepared by the ionic gelation using tripolyphosphatewere employed.40 A similar polymeric core coatedwith bovine serum albumin (BSA) serving as a controlnanovehicle (CNV) was also studied. The BBB uptakeof 125I-SNVs and 125I-CNVs was evaluated for thesematrices in mice. The uptake and transcytosis of SNVsand CNVs across the bovine brain microvascularendothelial cells (BBMECs) was evaluated using flowcytometry and confocal microscopy. Plasma clearanceof 125I-SNVs was nine times higher than that of125I- CNVs. However, the uptake of 125I-SNVs invarious brain regions was about 8 to 11 times higherthan that of 125I-CNVs. The uptake of fluoresceinisothiocyanate-bovine serum albumin (FITC-BSA)loaded SNVs in BBMECs was twice the uptakeof FITC-BSA loaded CNVs. Confocal micrographsdemonstrated the uptake and transcytosis of AlexaFluor 647 labeled SNVs, but not of CNVs across the

BBMEC monolayer. It was thus concluded SNVs arecapable of carrying a payload of model protein acrossthe BBB to target to the cerebral amyloid.

CONCLUSIONS

A century of research has passed since the discoveryand definition of AD, the most common dementingdisorder worldwide. However, the disease lacksdefinite diagnostic approaches and effective cure at thepresent. Moreover, the currently available diagnostictools are not sufficient for an early screening of ADin order to start preventive approaches. Recently theemerging field of nanotechnology and nanomedicinehas promised new ways to solve some of the ADchallenges. However, an important and long-term goalof pharmaceutical industry is to develop therapeuticagents that can be selectively delivered to specificsites of the human body to maximize therapeuticindex. At any rate, the ‘magic bullet’ concept, firsttheorized by Paul Ehrlich in 1891 represents theearly description of the drug-targeting paradigm. Theaim of drug targeting has been to deliver drugs tothe right place at the right concentration for theright time. Drug’s characteristics differ substantially inchemical composition, molecular size, hydrophilicityand protein binding, and the essential characteristicsthat identify its efficacy are highly complex. All ofthese are to be comprehensively investigated to bringnew compounds to the market even though onlya fraction of them would reach any active clinicalpractice. The use of nanotechnology for the purposeof diagnostics and imaging will soon become animportant tool in modern nanomedicine.

In the present study, in vivo detection of amy-loid plaques for the early diagnosis of AD has beendescribed. Presently, histological confirmation of theplaques and of the NFTs is the only definitive modeof diagnosis. This is true despite the fact that patientstypically have clinical diagnoses based on cognitivetests, medical histories, etc. However, non-invasivein vivo detection affords the patients an opportunityto receive the most appropriate patient care as early aspossible. Likewise, it allows clinicians and researchersthe prospect of monitoring disease progression andobjectively assessing the efficacy of therapeutic inter-ventions. Hence, healthcare professionals will be ableto design appropriate therapeutic strategies. Animalimaging studies will assist in drug developmentprocess and reduce the number of animals used indrug discovery process. However, the greatest benefit,perhaps, comes in the possibility of early detection andinitiation of appropriate therapeutic regimen, thereby



extending the patient’s life and improving his/her qual-ity of life. Successful plaque labeling and subsequentimaging in the animal model precipitates non-invasiveimaging in the intact living human brain. Polysorbate-coated NPs are thought to mimic the low-densitylipoproteins (LDL), allowing them to be transported

into the brain by the same endocytotic process asLDL undergoes at the BBB. Accumulating existingevidence suggests that the field of nanomedicine in thecontext of using polymeric NPs for delivering drug(s)to the brain represents the most novel approach innanoparticulate delivery technologies.

ACKNOWLEDGEMENTS

Tejraj M. Aminabhavi and Anjanapura V. Raghu thank the University Grants Commission, New Delhi, India(Grant No. F1-41/2001/CPP-II) for a major support to establish Center of Excellence in Polymer Sciencebetween 2002 and 2007. The authors thank Dr. Veera Arora for technical assistance and financial support ofDr. Jack Krohmer Professorship (FSB) is gratefully acknowledged.

REFERENCES

1. Torchilin VP. Recent advances with liposome as phar-maceutical carriers. Nature Rev Drug Discov 2005,2:145–160.

2. Duncan R. The dawning era of polymer therapeutics.Nature Drug Discov 2003, 2:347–360.

3. Boado RJ. Antisense drug delivery through the blood-brain barrier. Adv Drug Del Rev 1995, 15:73–107.

4. Roney CA, Kulkarni PV, Arora V, Antich P, Bonte F,et al. Targeted nanoparticles for drug delivery throughthe blood–brain barrier for Alzheimer’s disease. J Con-trol Release 2005, 108:193–214.

5. Gutman R, Peacock G, Lu D. Targeted drug deliveryfor brain cancer treatment. J Control Release 2000,65:31–41.

6. Zhuang Z. Radio-iodinated styrylbenzenes andthioflavins as probes for amyloid aggregates. J MedChem 2001, 44:1905–1914.

7. Smith, Q A review of blood brain barrier transporttechniques. The Blood Brain Barrier: Biology andResearch Protocols, ed. S Sag. Vol. 89. 2003, Totowa,NJ: Humana Press, Inc. 193–205.

8. Huang X. The AB peptide of Alzheimer’s diseasedirectly produces hydrogen peroxide through metalion reduction. Biochemistry 1999, 38:7609–7616.

9. Ritchie C. Metal-protein attenuation with iodochlorhy-dorxyquin (clioquinol) targeting AB amyloid depositionand toxicity in Alzheimer disease. Arch Neurol 2003,60:1685–1691.

10. Mundargi RC, Ramesh Babu V, Rangaswamy V,Patel P, Aminabhavi TM. Nano/micro technologies fordelivering macromolecular therapeutics using poly(d,l-lactide-co-glycolide) and its derivatives. J ControlRelease 2008, 125:193–209.

11. Wang G, Uludag H. Recent developments innanoparticle-based drug delivery and targeting systems

with emphasis on protein-based nanoparticles. ExpertOpin Drug Delivery 2008, 5:499–515.

12. Soppimath KS, Aminabhavi TM, Kulkarni AR,Rudzinski WE. Biodegradable polymeric nanoparti-cles as drug delivery devices. J Control Release 2001,70:1–20.

13. Gulyaev AE, Gelperina SE, Skidan IN, Antropov AS,Kivman GY, et al. Significant transport of doxorubicininto the brain with polysorbate 80–coated nanoparti-cles. Pharm Res 1999, 16:1564–1569.

14. Hartig W, Paulke BR, Varga C, Seeger J, HarkanyT, et al. Electron microscopic analysis of nanopar-ticles delivering thioflavin-T after intrahippocampalinjection in mouse: implications for targeting beta-amyloid in Alzheimer’s disease. Neurosci Lett 2003,338:174–176.

15. Siegemund T, Paulke BR, Schmiedel H, Bordag N,Hoffmann A, et al. Thioflavins released from nanopar-ticles target fibrillar amyloid beta in the hippocampusof APP/PS1 transgenic mice. Int J Dev Neurosci 2006,24:195–201.

16. Barnabas W, Kumar SM, Kumaraswamy S,Kokilampal PSK, Nallupillai P, et al. Poly(n-butylcyanoacrylate) nanoparticles coated with polysor-bate 80 for the targeted delivery of rivastigmine intothe brain to treat Alzheimer’s disease. Brain Res 2008,1200:159–168.

17. Wilson B, Samanta MK, Santhi K, Kumar KPS,Paramakrishnan N, et al. Targeted delivery of tacrineinto the brain with polysorbate 80-coated poly(n-butylcyanoacrylate) nanoparticles. Eur J Pharm Bio-pharm 2008, 70:75–84.

18. Lockman PR, Mumper RJ, Khan MA, Allen DD.Nanoparticle technology for drug delivery across theblood–brain barrier. Drug Dev Ind Pharm 2002,28:1–13.



19. Calvo P, Gouritin V, Chacun H, Desmaele D,D’Angelo J, et al. Long-circulating PEGylated poly-cyanoacrylate nanoparticles as new drug carrier forbrain delivery. Pharm Res 2001, 18:1157–1166.

20. Reddy JS, Venkateswarlu V. Novel delivery systemsfor drug targeting to the brain. Drugs Future 2004,29:63–69.

21. Kreuter J. Influence of the surface properties onnanoparticle-mediated transport of drugs to the brain.J Nanosci Nanotechnol 2004, 4:484–488.

22. Koo YEL, Reddy GR, Bhojani M, Schneider R,Philbert MA, et al. Brain cancer diagnosis and ther-apy with nanoplatforms. Adv Drug Deliv Rev 2006,58:1556–1577.

23. Lovell M. Copper, iron and zinc in Alzheimer’s diseasesenile plaques. J Neurol Sci 1998, 158:47–52.

24. Suh SW. Histological evidence implicating zinc inAlzheimer’s disease. Brain Res 2000, 852:274–278.

25. Cherney RA. Treatment with a copper-zinc chelatormarkedly and ragidly inhibits B-amyloid accumulationin Alzheimer’s disease transgenic mice. Neuron 2001,30:665–676.

26. Yassin MS. Changes in uptake of vitamin B (12) andtrace metals in brains of mice treated with clioquinol.J Neurol Sci 2000, 173:40–44.

27. Bush AI. Metal complexing agents as therapiesfor Alzheimer’s disease. Neurobiol. Aging 2002,23:1031–1038.

28. Kontush A. Amyloid-beta is an antioxidant for lipopro-teins in cerebrospinal fluid and plasma. Free Radic BiolMed 2001, 30:119–128.

29. Teixido M, Giralt E. The role of peptides in blood-brain barrier nanotechnology. J Peptide Sci 2008,14:163–173.

30. De Boer AG, Gaillard PJ. Strategies to improve drugdelivery across the blood-brain barrier. Clin Pharma-cokinetics 2007, 46:553–576.

31. Kreuter J, Alyautdin RN, Kharkevich DA, Ivanov AA.Passage of peptides through the blood brain barrier

with collodial polymer particles (nanoparticles). BrainRes 1995, 674:171–174.

32. Klunk WE. Uncharged thioflavin-T derivatives bind toamyloid-beta protein with high affinity and readilyenter the brain. Life Sci 2001, 69:1471–1484.

33. Ahi J, Radulovic J, Spiess J. The role of hippocampalsignaling cascades in consolidation of fear memory.Behav Brain Res 2004, 149:17–31.

34. Blank T. Priming of long-term potentiation in mousehippocampus by corticotropin-releasing factor andacute stress: implications for hippocampus-dependentlearning. J Neurosci 2002, 22:3788–3794.

35. Opazzo C, Luza S, Villemagne VL, Volitakis I, RoweC, et al. Radio-ioiodinated clioquinol as a biomarkerfor β-amyloid: Zn2+ complexes in Alzheimer’s disease.Aging Cell 2006, 5:69–79.

36. Agdeppa ED, Kepe V, Liu J, Small GW, Huang SC,et al. 2-Dialkylaminoa-6-acylmalononitrile substitutednaphthelnes (DDNP analog): novel diagnostic and ther-apeutic tools in Alzheimer’s disease. Mol Imaging Biol2003, 5:404–417.

37. Newberg AB, Wintering NA, Plossi K, Hochold J,Stabin MG, et al. Safety and biodistribution anddosimetry of 123I-IMP: A novel Amyloid plaque-Imaging Agent for the Diagnosis of Alzheimer’s Disease.J Nucl Med 2006, 47:748–754.

38. Maurice T, Lockhart BP, Privat A. Amnesia inducedin mice by centrally administered β-amyloid pep-tides involves cholinergic dysfunction. Brain Res 1996,706:181–193.

39. Cattaneo A, Capsoni S, Paoletti F. Towards non inva-sive nerve growth factor therapies for Alzheimer’sdisease. J Alzheimers Dis 2008, 15:255–283.

40. Agyare EK, Curran GL, Ramakrishnan M, Yu CC,Poduslo JF, et al. Development of a smart nano-vehicleto target cerebrovascular amyloid deposits and brainparenchymal plaques observed in Alzheimer’s diseaseand cerebral amyloid angiopathy. Pharm Res 2008,25:2674–2684.

FURTHER READING

Roney C, Kulkarni PV, Arora V, Antich P, Bonte F, et al. Targeted nanoparticles for drug delivery through theblood–brain barrier for Alzheimer’s disease. J Control Release 2005, 108: 193–214.

Roney C. PhD thesis, UT Southwestern Medical Center, Dallas, TX, 2006.


Quinoline-n-butylcyanoacrylate-based nanoparticles for brain targeting for the diagnosis of Alzheimer's disease

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