Lactoferrin-Conjugated Dendritic Nanoconstructs for LungTargeting of Methotrexate

BALAK D. KURMI, VIRENDRA GAJBHIYE, JITENDRA KAYAT, NARENDRA K. JAIN

Department of Pharmaceutical Sciences, Pharmaceutics Research Laboratory, Dr. Hari Singh Gour University, Sagar,Madhya Pradesh 470003, India

Received 19 June 2010; revised 19 October 2010; accepted 12 December 2010

Published online 14 January 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22469

ABSTRACT: The present investigation was aimed at developing and exploring the potential oflactoferrin (Lf)-conjugated dendritic nanocomposite for lung targeting of methotrexate (MTX).The 5.0G poly(propylene imine) (PPI) dendrimer and Lf-conjugated 5.0G PPI dendrimer weresynthesized and characterized by Fourier-transform infrared spectroscopy, nuclear magneticresonance, and transmission electron microscopy . The entrapment efficiency, in vitro release,and hemolytic toxicity were assessed. Pharmacokinetic and organ distribution studies were car-ried out to evaluate in vivo targeting potential of developed system. The pharmacokinetic studiesshowed that elimination half-life of MTX-loaded plain PPI dendrimer (10.41 ± 2.12 h, p < 0.05)and MTX-loaded Lf-conjugated PPI dendrimer (12.23 ± 1.53 h, p <0.01) was significantly higherthan the free drug (5.85 ± 1.19 h). Organ distribution assessment of different formulations dis-played significant (p <0.05) higher accumulation of drug in lungs by MTX–Lf–PPI (1329 ± 26.7ng/g of tissue) as compared with MTX–PPI (721 ± 23.4 ng/g of tissue) and free MTX (575 ± 19.7ng/g of tissue) after 6 h of administration. The result suggested that Lf-conjugated 5.0G PPIdendrimer-based formulations to be approximately 1.5 times and 2.5 times superior to plain5.0G PPI dendrimer as well as pure MTX, respectively, for lung targeting of anticancer drugs.© 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:2311–2320,2011Keywords: dendrimers; methotrexate; lactoferrin; lung targeting; nanoconstructs; pul-monary drug delivery; cancer chemotherapy; drug targeting; macromolecular drug delivery

INTRODUCTION

Lung cancer is the major cause of cancer mortalityworldwide. It is still a serious malignancy in manycases due to the lack of major advancements in treat-ment strategy.1 Current treatments for lung cancerhave shown little success because they cannot curedisseminated tumors with an acceptable level of tox-icity. Thus, one alternative strategy that has shownpromise in the treatment of lung cancer is targetedtherapy.2 Targeted therapy was conceptualized asa means of exploiting specific molecular alterationsassociated with cancers in order to selectively killtransformed cells and spare normal healthy tissues.Targeted therapies are anticipated to have fewerassociated toxicities than standard chemotherapies,which rely predominately on increased rates of cell di-vision to enhance killing of the tumor cells comparedwith healthy tissues. For tumors that are treated with

Correspondence to: Narendra K. Jain (Telephone: +91-9329041469; Fax: +91-7582404813; E-mail: jnarendr@yahoo.co.in)Journal of Pharmaceutical Sciences, Vol. 100, 2311–2320 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

radiation and/or surgery, systemically delivered tar-geted therapies also have the potential to eliminatemicrometastases that might not be eliminated withradiation therapy (RT) and/or surgery.3

Drug delivery to lungs appears to be an attrac-tive proposition on account of large surface area ofalveolar region. It provides tremendous opportunitiesto improve drug therapies using novel drug deliverysystems. Lower thickness of the epithelial barrier, ex-tensive vascularization, relatively low proteolytic ac-tivity in the alveolar space as compared with otherroutes of administration, and absence of the first-passmetabolism facilitate enhanced systemic availabilityof the active medicament.4 Ranney5 demonstrated theimportance of the targeted drug delivery to the lungsand pointed out that diseases located in this regionlend themselves to a drug-targeting approach becauseof ready access to these organs by both the intra-venous (i.v.) and intratracheal routes. Carrier systemsplay an important role in the targeting of drug(s),proteins, and peptides. Carriers could supplementsustained drug delivery to the lungs, extend du-ration of action, reduce therapeutic dose, improve

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2312 KURMI ET AL.

patient compliance, and reduce adverse effects ofhighly toxic drugs.6 The targeting of drugs to thelungs can deliver therapeutic agents to the diseasedregions by reducing their distribution to the non-target organs.7

Lactoferrin (Lf) is a nonheme iron-binding proteinthat is part of the transferrin protein family, alongwith serum transferrin, ovatransferrin, melanotrans-ferrin and the inhibitor of carbonic anhydrase,8 andcationic in nature.9 Lf was investigated as a target-ing ligand for receptor-mediated gene delivery to hu-man bronchial epithelial cells. A high number of Lfreceptors (LfRs) were detected on bronchial epithe-lial cells (BEAS-2B) but not on alveolar epithelial(A549) cells by fluorescence microscopy and FACSmeasurements, suggesting potential targeting selec-tivity for bronchial epithelial cells. Previous studieshave demonstrated that LfRs are expressed on theapical surface of bronchial epithelial cells.10 Thus, Lfmay serve as a suitable targeting ligand for receptor-mediated delivery to the lung for treating specific lungcancers originating from bronchioles (Fig. 1), for ex-ample, nonsmall cell lung carcinoma, which usuallystarts near a central bronchus or in peripheral lungtissue.

Dendrimers are unimolecular polymeric systemssynthesized in a reiterative manner. At the sametime, their synthesis can be so optimized as to controltheir size, shape, molecular mass, composition, andreactivity.11 Dendrimers have hyperbranched struc-ture with precisely placed functional groups that bearimportant role in controlling the properties of ther-apeutic moieties that are encapsulated or complexed

with it.12,13 Most eminent properties of dendrimer areits monodispersive nature, globular shape, and highlycontrolled architecture, which also makes them effi-cient carrier system for drugs.14,15 In recent times,many advancements have occurred in the utiliza-tion of dendrimers to treat cancer, including theiruse as delivery systems for potent anticancer drugs.16

The present study was aimed at developing and ex-ploring the Lf-conjugated Poly(propylene imine) (PPI)5.0G dendrimers for targeting to lungs. Methotrexate(MTX) was taken as a model anticancer drug.

EXPERIMENTS

Materials

Methotrexate hydrate was a benevolent gift from M/sKhandelwal Laboratories, (Mumbai, Maharashtra,India), and Lf was a kind gift from Dr. T.P. Singh,All India Institute of Medical Sciences (New Delhi,India). Ethylene diamine, acrylonitrile, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) werepurchased from CDH (New Delhi, India). Cellulosedialysis bag (MWCO 6–7 kDa), was purchased fromSigma, whereas Reney Nickel was purchased fromMerck India. All other chemicals were of reagentgrade and purchased from CDH.

Synthesis of 5.0GPPI Dendrimer

Poly(propylene imine) dendrimers (5.0G) were syn-thesized by divergent method reported earlier.17,18

Briefly, double Michael addition reaction method wasused to produce half generation (-CN terminated) by

N

N

NN

NN

N

N

N

NN

H2N NH2H2NNH2

N N

NH2NH

NH2

NH2

N

NN

NN

NH2NH2

NHNH2

NN

NH2NH2NH2NH2

N N

N

N

NNH2

NH2

NH2

NH2NN

NH2

NH2

NH2

NH2

N

NN

NN

H2NH2N

H2N H2N

N N

H2NH2N

H2NNH

NN

N

N

N

H2N

H2N

H2N

H2N N

NH2N

H2N

H2NH2N

NN

N

N

N

NH2

NH2

NH2

NH2N

N NH2

NH2

NH2

NH2

N

N

NNN

H2NH2N

H2NNH

N N

H2NH2N NH2H2N

NN

N

NN

H2N

H2NH2N

H2N

NN

H2N

H2N

H2NH2N

C

O

C

O

CO

CO

Lactoferrin receptor

Lactoferrin

Lactoferrin receptorbinding site

Poly (propylene imine)dendrimer

N

N

N

N

H2N

NH2

CH2N

CONH

C

COOH

CH2CH2COOHH

H3C

N

NN

NH2N

NH2

CH2N CONH

C COOHHOOCH2CH2C

H

CH3

N

N

NN

H2NNH2

CH

2N

CO

NH

C

COOH

HOOCH2CH2C

H

H3C

N

NN

NH2N

NH2

CH2N CONH

C COOHHOOCH2CH2C

H

CH3

N

N

N N

NH2

H2N

CH

2 NC

ON

HC

COOH

CH2CH2COOH

H

CH3

NN

N

N

NH2

H2N

CH

2 N

CO

NH

C

COOH

HOOCH2CH2C

H

CH3

N

N

N

N

H2N

NH2

CH 2

N

CO

NH

C

COOH

HOOCH2CH2C

H

H3CN

N

N

N

H2N

NH2

CH2N

CONH

C

HOOC

CH2CH2COOH

H

H3C

Methotrexate

Bro

nchi

al e

pith

elia

l cel

l

Figure 1. Schematic diagram of methotrexate-loaded Lactoferrin-Conjugated dendrimer fortargeting lactoferrin receptors in bronchial epithelial cells.

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adding acrylonitrile to the initiator core, ethylenedi-amine (EDA). It was pursued by heterogeneous hy-drogenation using Reney Nickel catalyst to synthesize1.0G generation (-NH2) dendrimers. The reaction se-quence was repeated intermittently to fabricate PPIdendrimers up to fifth generation (PPI 5.0G). FTIRspectroscopy was carried out using PerkinElmer IRspectroscope. 1H NMR spectroscopy of the dendrimersample was carried out at 300 MHz in CDCl3 (BrukerDRX).

Synthesis and Characterization ofLactoferrin–Dendrimer Conjugate

Conjugation of 5.0G PPI dendrimer formulation withLf was performed by using carbidiimide chemistry,that is, coupling of carboxylic group of Lf with theterminal amino group of 5.0G PPI dendrimer in thepresence of 1-ethyl-3-(3-dimethylaminepropyle) car-bodiimide (EDC) in phosphate buffer (pH 5.0). Forthe preparation of Lf-conjugated 5.0G PPI dendrimer,a solution of Lf (2.0 mM) in phosphate buffer (pH5.0) and 10 mg of EDC was taken in a conical flaskcontaining phosphate buffer (pH 5.0) and stirred for30 min at room temperature. PPI dendrimer (1.0 mM)was added to above solution and continuously stirredfor 96 h at room temperature, followed by removal ofsolvent in a rotary vacuum evaporator19,20 and dia-lyzed through dialysis membrane [Molecular weightcut-off (MWCO) 100 kDa, Spectra/Por R©] to removeunconjugated Lf and 5.0G PPI dendrimers. The Lf-conjugated dendrimeric system was characterized byFTIR, 1H NMR, and MASS. Transmission electronmicroscopy (TEM) of lactoferrin–dendrimer conjugate(Lf–PPI) was performed to characterize dendrimers interms of their size. TEM was performed with the helpof transmission electron microscope (Morgani 268D,the Netherlands) after drying dendrimer solution oncarbon grid and staining negatively by 1% PTA (phos-photungustic acid) by metal shadowing techniques atElectron Microscopy Division, All India Institute ofMedical Sciences, New Delhi, India.

Encapsulation of MTX in Lf–PPI and Plain 5.0g PPIDendrimer

The known molar concentrations of PPI 5.0G den-drimer and Lf–PPI were dissolved separately in10.0 mL of PBS (pH 7.4) and mixed with 100.0 mgof MTX. This was continuously stirred for 3 days atroom temperature. The sample was dialyzed usingdialysis membrane (MWCO 1 kDa, Sigma) for 15 minto remove free drug from the formulations, which wasthen estimated spectrophotometrically (λmax 258 nm;UV-1601; Shimadzu, Kyoto, Japan) to determine in-directly the amount of drug loaded within the system.The dialyzed formulations were lyophilized and usedfor further characterization. All the experiments re-lated to MTX were carried out in dark.

Characterization of the MTX-LoadedLactoferrin–Dendrimer Conjugate System

Methotrexate-loaded lactoferrin–dendrimer conju-gate (MTX–Lf–PPI) and physical mixture of MTXand lactoferrin–dendrimer conjugate (MTX + Lf–PPI)were analyzed by infrared spectroscopic technique us-ing Nujol’s mull method at SAIF, Punjab University,Chandigarh in a PerkinElmer Spectrum RX-I FTIRSpectrophotometer.

Hemolytic Study

Whole human blood was collected from blood bankusing heparin as anticoagulant. This was centrifugedat 3000 rpm for 15 min, red blood cells (RBCs) sep-arated at the bottom of tube were washed with nor-mal saline to get a clear, colorless supernatant abovethe cell mass. The cells were resuspended in normalsaline to get 5% (w/v) suspension and were furtherused for hemolytic study.

One milliliter volume of the RBCs suspension wasadded to 1 mL of solution (0.01, 0.02, 0.03, 0.04, and0.05%, w/v) of plain 5.0G PPI dendrimer, free MTX,drug (MTX)-loaded 5.0G PPI dendrimer, Lf-5.0G PPIdendrimer conjugate, and drug (MTX)-loaded Lf-5.0GPPI dendrimer conjugate. One milliliter of RBCs sus-pension was added to 1 mL of water and 1 mL ofnormal saline to obtain 100% hemolysis and blanksamples, respectively. These tubes were shaken in-termittently and allowed to stand for 2 h. All thepreparations were centrifuged at 3000 rpm for 10 min.Supernatant was removed and after proper dilutionwith normal saline the absorbance was taken at λmax540 nm. The percentage hemolysis was calculated foreach sample by considering that 100% hemolysis oc-curred in water.17,18,21

In Vitro Release

The in vitro drug release characterization of the syn-thesized formulations involved assessment of theirdrug holding capacity and ability to release the samein the release media. The in vitro drug release fromdifferent formulations was carried out using dialysistube diffusion technique. Benzoylated dialysis mem-brane (MWCO 6000–7000 Da, Sigma) was selected forthe study; 2 mL solution of the formulation was placedin the dialysis sac, hermetically tied and immediatelysuspended in 50 mL of aqueous receptor medium. Thein vitro drug release experiments were performed inPBS (pH 7.4) under sink condition in the receptorcompartment through constant stirring using mag-netic stirrer (2 EMS, Remi equipments, New Delhi,India) at room temperature. The drug (MTX) releasewas determined in 1 mL of aliquot, after appropriatedilutions, spectrophotometrically at 258 nm. The vol-ume of sink receptor compartment was maintainedby replenishing with 1 mL of fresh sink solution

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after each withdrawal of 1 mL of the sample fromthe receptor medium.

In Vivo Studies

in vivo pharmacokinetic and organ distribution stud-ies were performed to evaluate fate of the developedformulation and lung targeting efficiency of developedformulation in rat model. All experiments were car-ried out in accordance with the protocol approved bythe Animal Ethical committee of Dr. H. S. Gour Uni-versity, Sagar, Madhya Pradesh, India. Separate an-imals were used for pharmacokinetic and organ dis-tribution studies.

Blood Level Studies

Sprague Dawley albino rats (2 months old, 100–150 g)of either sex obtained from Dr. H. S. Gour Univer-sity animal house were divided into three groupshaving three rats per group. Animals were fastedovernight before administration of dose. First groupwas administered free drug in normal saline throughi.v. route via tail vein, whereas second and thirdgroups received drug-loaded plain PPI dendrimer(MTX–PPI) and drug-loaded Lf-conjugated PPI den-drimer (MTX–Lf–PPI), respectively, suspended innormal saline. All the three groups were administeredMTX in a dose of 20mg/kg body weight.

Following the administration, blood samples(0.1 mL each time) were collected from retro-orbitalplexus at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, 18, 24, 36, and 48 h.In each case, blood was centrifuged at 3000 rpm for10 min to separate RBCs and serum. The upper su-pernatant (serum) was collected separately with thehelp of micropipette and MTX was estimated at dif-ferent time intervals by a reported high-performanceliquid chromatography (HPLC) method22 employinga C18 column of 150 × 4.5 mm internal diameter and5 :m particle size (Thermo Hypersil, ODS) protectedby a guard column. The column effluent was moni-tored by a UV detector at 254 nm. The elution wascarried out by gradient elution technique and the mo-bile phase consist of TRIS–phosphate buffer (pH 5.7):methanol: acetonitrile (82:11:7) with a flow rate of1.5 mL/min. The retention time for MTX was foundto be 4.3 min. The pharmacokinetic parameters werecalculated taking help of “PK-analyst,” scientific soft-ware from Micromath Inc. (UK).

Organ Distribution Studies

Albino rats of either sex were divided into threegroups having three rats per group. Animals werefasted overnight before administration of dose. PlainMTX, MTX–PPI, and MTX–Lf–PPI were adminis-tered through tail vein to each rat of first, second,and third group, respectively. Rats of each group weresacrificed 6 h post-administration. Different organs(liver, kidney, lung, and spleen) were isolated and

dried with tissue paper and weighed. Organs wereadded with required quantity of ethanol and homog-enized well using tissue homogenizer (York Scien-tific Instrument, New Delhi, India). The homogenateswere centrifuged at 5000 rpm for 15 min and clearsupernatant was collected in vials and analyzed forMTX by HPLC.

Statistical Analysis

The results are expressed as mean ± standard de-viation (S.D.; n = 3) and statistical analysis wasperformed with SPSS 10.1 for Windows R© (SPSS R©,Chicago, Illinois). The differences between the Lf–PPIdendrimers and plain PPI dendrimers were observedby pair-wise comparisons using unpaired t-test per-formed in GraphPad InStat version 3.00 for Windows95 (GraphPad Software, San Diego, California).

RESULTS

Synthesis and Characterization of 5.0g PPI Dendrimers

The PPI dendrimers were synthesized by repetitionof double Michael addition to acrylonitrile and sub-sequent hydrogenation (reduction) to yield primaryamine terminated generations. EDA was used asinitiator core.

Synthesis of 0.5G PPI dendrimer was confirmed byFTIR spectroscopy. Strong peak of nitrile at 2248.1cm−1 and CH2 bending at 1463.9 cm−1 confirmedsynthesis of 0.5G dendrimer. FTIR spectra of 5.0GPPI dendrimer showed C-C bending of alkane (1106.6cm−1), C-C stretch of alkane (1410.4 cm−1), N-H bend-ing vibrations (1564.2 cm−1), C-H stretch (2966.4cm−1), and weak peak of C≡N stretch of nitrile(2251.3). Furthermore, N-H stretch of primary amineat 3409.8 cm−1 confirmed that most of the nitrile ter-minal dendrimers were converted to primary amineterminal dendrimers.

The synthesis of 5.0G PPI dendrimer was furtherconfirmed by the obtained peaks and shifts in 1HNMR. Peaks of alkane were obtained between 0.8and 1.2 ppm and those of alkyl amine between 1.6and 1.75 ppm. Alkyl amine peaks were exhibited be-tween 2.7 and 2.9 ppm, amine peaks were at 2.3 ppmand tertiary amine peaks between 2.4 and 2.6 ppm.Results were similar to those reported by us for thesynthesis of PPI dendrimers.17,18

Synthesis and Characterization of Lf–PPI)

The conjugation of Lf with 5.0G PPI dendrimer wasperformed via carbodiimide chemistry, wherein car-boxylate group of Lf was conjugated with terminalamino group of 5.0G PPI dendrimer with the help ofEDC as coupling agent. The FTIR analysis of Lf–PPIsystem showed the peak of C=O stretch of amidelinkage (CO-NH) at 1652.0 cm−1 formed between the

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Figure 2. Fourier-transform infrared spectrum of lactoferrin–dendrimer conjugates (Lf–PPI).

carboxylate group of Lf with the amine group of 5.0GPPI dendrimer. Strong bands at 3420.7 cm−1 sug-gested the free terminal amine groups of dendrimer(Fig. 2).

NMR spectroscopy further confirmed conjugationof Lf with PPI dendrimer. Shift at 7.8 ppm helps todistinguish the -CONH amide group of conjugatedsystem. Further shift at 2.6 ppm depicts the pres-ence of CH2-NH2 groups (Fig. 3). The TEM of Lf–PPIs displayed nanometric sized units. The increasein particle size on macromolecular conjugation wasalso observed (Fig. 4). MASS spectroscopy suggestedthat approximately three molecules of Lf were conju-gated on surface amine groups of one PPI dendrimermolecule.

Characterization of MTX-Lf-PPI

Entrapment of drug (MTX) was confirmed by FTIRspectroscopy of MTX-loaded lactoferrin–dendrimerconjugates (MTX–Lf–PPI) and the infrared spectrumof physical mixture of MTX and lactoferrin–den-drimer conjugate. These spectra closely resembledeach other, which suggested physical entrapment ofdrug (MTX) in Lf–PPI and ruled out any chemicalalteration (Fig. 5).

Drug Entrapment Efficiency

The entrapment efficiency of the system was calcu-lated as a ratio of amount of drug entrapped by thesystem to the amount of drug taken for drug load-ing expressed as a percentage. MTX loading in theplain 5.0G PPI dendrimers and Lf-conjugated 5.0 G

PPI dendrimers were found to be 69.2 ± 3.6% and76.3 ± 4.1%, respectively (n = 3). The data depictshigher drug (MTX) entrapment inside the conjugateddendritic systems compared with plain 5.0G PPI den-drimers. The drug entrapment for the systems thushad increased with conjugation of Lf to PPI. But thedifference between drug loading was not found statis-tically significant (p = 0.08).

Hemolytic Study

Among the systems under study, PPI was found topossess maximum hemolytic activity, whereas Lf–PPIwas found to possess negligible hemolytic activity.In all the cases, hemolytic activity was found to beconcentration-dependent. Although PPI was found topossess high hemolytic activity (3.53 ± 0.175) at a con-centration of 0.05%, Lf–PPI (1.54 ± 0.42) was foundto possess very little hemolytic activity. The resultsof hemolytic toxicity of PPI matched with the earlierreports from our laboratory.17,18 The hemolytic toxi-city of free MTX was higher at all concentration ascompared with plain PPI, Lf–PPI, and Lf–PPI–MTX,but it was lower than PPI–MTX. Conjugation of Lf toPPI drastically reduced the hemolytic activity due tomasking of cationic terminal amino groups responsi-ble for the hemolytic activity of PPI (Fig. 6).

In Vitro Release

The rate of drug release was studied in PBS (pH 7.4)and suggested controlled-release drug delivery be-cause of the ability of dendrimer to solubilize the

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Figure 3. Nuclear magnetic resonance spectrum of Lactoferrin-Conjugated PPI 5.0 Gdendrimer.

hydrophobic drug in their interior, and to release itslowly. The results of in vitro release of MTX from PPIand Lf–PPI are presented in Figure 7. The in vitrodrug release profile showed that there was signifi-cant (p = 0.0005) decline in release rate of MTX from

Lf–PPI as compared with PPI. The plain PPI com-pletely releases the drug up to 76 h, whereas the PPI-based nano containers (Lf–PPI) prolonged the releaserate up to 88 h (96.0 ± 2.4%). The free MTX was re-leased almost completely within 2 h.

Figure 4. Transmission electron microscopy of Lactoferrin-Conjugated 5.0G PPI dendrimer.

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Figure 5. Fourier-transform infrared spectrum of(a) MTX, (b) MTX-loaded lactoferrin–dendrimer conjugate(MTX–Lf–PPI), and (c) physical mixture of MTX and lacto-ferrin–dendrimer conjugate (MTX + Lf–PPI).

In Vivo Studies

Blood Level Studies

Blood level studies of plain drug and dendrimer for-mulations were assessed to determine the releaseand performance of formulations in vivo. The for-mulations were predialyzed to remove unentrappeddrug and drug concentration equivalent to that offree drug was administered to the rats by i.v. route.The blood plasma was used to determine the con-

Figure 6. Percent hemolysis from different dendrimer for-mulations (n = 3).

centration of drug in blood samples at various timeintervals. The concentration of MTX in plasma, af-ter i.v. administration of free MTX, was found to be5309 ± 34.8 ng/mL after 0.5 h that decreased to 79± 8.3 ng/mL after 36 h (Fig. 8). This may be due tothe excretion of drug from kidney and its simultane-ous tissue distribution in various organs. However,the concentration of drug in plasma after administra-tion of different dendrimer formulations (MTX–PPIand MTX–Lf–PPI) was considerably low. In case ofMTX–PPI formulation, the maximum concentrationrecovered in serum was found to be 3508 ± 28.5 ngafter 6 h (Fig. 8), which was decreased to 214 ± 13.6 ngafter 48 h, whereas in case of MTX–Lf–PPI formula-tion, the maximum concentration recovered in serumwas found to be 3879 ± 35.6 ng after 5 h, which was de-creased to 339 ± 14.8 ng after 48 h (Fig. 8). The bloodlevel studies of dendrimeric formulations showedin vivo sustained release of entrapped MTX. The

Figure 7. Cumulative percent drug release from den-drimer formulations (n = 3).

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Figure 8. Blood plasma level studies of MTX afterintravenous administration from dendrimer formulations(n = 3).

significant reduction in drug concentration in serumin case of PPI dendrimer formulation as comparedwith free drug may be attributed to the fact that mostof the drug present in blood was entrapped in PPIdendrimer which led to reduced toxicity of MTX. Phar-macokinetic parameters showed increased t1/2 and Vd,whereas decreased elimination rate remains constantin case of MTX–Lf–PPI and MTX–PPI as comparedwith free drug (Table 1).

Biodistribution (Organ Distribution) Studies

Estimation of drug concentration in liver, lung, kid-ney, and spleen after i.v. administration of free MTX at

Table 1. Pharmacokinetic Parameters of MTX AfterIntravenous (i.v.) Administration from Dendrimers (n=3)

Pharmacokinetic Parameters

Systems Cmax (ng/mL) t1/2 (h) Vd (L/kg) Kel (h -1)

Plain MTX 5309 ± 34.8 5.85 0.298 0.1185MTX–PPI 3508 ± 28.5 10.41∗ 1.22 0.0666MTX–Lf–PPI 3879 ± 35.6 12.23∗ 1.75 0.0567

∗p < 0.001MTX, methotrexate; PPI, poly(propylene imine); Lf, lactoferrin.

6 h time interval showed maximum accumulation ofthe drug in non-target organs. The maximum amountof drug accumulated in different organs following i.v.administration of free drug after 6 h were found to be1039 ± 27.2 ng/g of tissue in liver, 789 ± 16.9 ng/g oftissue in kidney, 575 ± 19.7 ng/g of tissue in lung oftissue, and 686 ± 19.7 ng/g of tissue in spleen (Fig. 9).In case of MTX–PPI, the liver uptake was found to be1275 ± 30.7 ng/g of tissue after 6 h, whereas in case ofMTX–Lf–PPI formulation, the concentration of drugrecovered by the liver was found to be 927 ± 21.4 ng/gof tissue after 6 h.

The drug concentration recovered from the lungsin case of Lf-conjugated formulation (1329 ± 26.7 ng/g of tissue) was significantly higher than free MTX(575 ± 19.7 ng/g of tissue, p = 0.001) and MTX–PPIformulation (721 ± 23.4 ng/g of tissue, p = 0.001) in6 h (Fig. 9). The results show that there was approx-imately 2.5-fold and 1.5-fold increase in lung uptakein case of Lf-conjugated system as compared with freedrug and plain dendrimer formulations.

Figure 9. Organ distribution of MTX after intravenous administration from dendrimers after6 h (n = 3).

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DISCUSSION

In the present study, lung targeting potential of Lf-conjugated dendrimers was explored. 5.0G PPI den-drimers and Lf-conjugated 5.0G PPI dendrimer weresynthesized and characterized by FTIR, 1H NMR,and TEM microscopic means. The reports of analysisof PPI were in accordance with earlier reports.17,18

The existence of an amide (CO-NH) at 1652.0 cm−1

in FTIR spectra of Lf–PPI (Fig. 2) might be at-tributed to amide bond formation between the car-boxylate groups of Lf with the terminal amino groupof 5.0G PPI dendrimer. The NMR spectroscopy shiftat 7.8 ppm also helped to distinguish the CO-NHamide group of conjugated system (Fig. 3). The drugloading in different dendrimeric systems was accom-plished by excess drug interaction for prolonged dura-tion with the dendrimeric system. Dendrimers with ahydrophobic interior and hydrophilic chain ends areable to solubilize hydrophobic compounds in aqueoussolutions; hence, the MTX molecules must be phys-ically entrapped in the interior of dendrimers. Thismay be attributed to the larger PPI dendrimer in-terior of Lf-conjugated dendrimers, which accountsthe higher encapsulation ability. The higher encapsu-lation ability of the modified dendrimer was due tolong chain length of the Lf attached to the dendrimermoiety. The similar FTIR spectrum of physical mix-ture of PPI and MTX and MTX-loaded PPI suggestedphysical entrapment of MTX in PPI and Lf–PPI den-drimers. The hemolytic toxicity of PPI is due to thepolycationic nature due to the primary amine groupspresented on the surface of PPI dendrimers.11 It isof great importance to note that the cytotoxicity ofPPI could be significantly reduced through Lf cou-pling. The results suggested that this may be primar-ily through shielding of parts of the PPI dendrimerswith Lf that could result in the reduction of primaryamine groups presented on the PPI dendrimers sur-face that are responsible for hemolysis. The in vitrorelease studies revealed that release rate of conju-gated 5.0G PPI dendrimer was found to decrease dueto closed structure and higher steric resistance offeredby attached groups.

Blood level studies revealed highest Cmax for freeMTX at a very early stage (0.5 h), as the drug wasavailable in the blood immediately after i.v. adminis-tration. On the contrary, Cmax, in case of plain PPI andLf–PPI was achieved at fifth and sixth h, respectively.The reason behind higher time taken for Cmax by den-drimer formulation is that the drug has to leach out ofdendritic system to be available in the blood. Pharma-cokinetic studies showed that Lf–PPI increased thehalf-life and decreased the elimination rate of MTX.The data suggested that the Lf–PPI prolonged the ac-tion of drug by sustaining the release of the entrappeddrug (Table 1). Lf–PPI was found to target higher con-

centration of MTX in lungs cells as evident from thefact that the accumulation of MTX–Lf–PPI was ap-proximately 2.5 times higher than that of free drugand 1.5 times higher than that of MTX–PPI. The in-creased uptake of conjugated dendrimer by lung ascompared with plain drug and unconjugated formu-lation may be due to the Lf recognition by the cellmembrane of bronchial epithelial cells of the lung tis-sue. The Lf–PPI might have interacted with the LfRspresent on the surface of alveolar bronchial epithe-lial cells, which led to its enhanced uptake.19 Hencemany fold increase in uptake of MTX was achievedby Lf-conjugated PPI dendrimer formulation as com-pared with unconjugated PPI dendrimer formulationand free drug solution due to the presence of LfRs onthe surface of bronchial epithelial cell of lung.

CONCLUSION

The in vitro drug release studies, in vivo pharma-cokinetic, and tissue distribution studies suggestedthat in the treatment of lung cancer, Lf-conjugated5.0G PPI dendrimers can be a promising drug target-ing carrier for anticancer bioactive. Encapsulation ofMTX in Lf-conjugated 5.0G PPI dendrimer enhancesthe residence time as well as concentration of drugin lung, which could be useful in reducing the dosingfrequency as well as dose. The conjugated dendrimeralso showed less hemolytic toxicity. This could helpin the reduction of toxicity normally associated withanticancer bioactives. Present study suggests that Lfcan target MTX encapsulated 5.0G PPI dendrimer tothe lung. Hence Lf mediated biodisposition and cel-lular interaction of 5.0G PPI dendrimer, especially atthe target sites would be a focal paradigm for theupcoming research in the field of lung cancer drugdelivery.

ACKNOWLEDGMENTS

Balak D. Kurmi would like to acknowledge All IndiaCouncil of Technical Education, New Delhi, India, forproviding financial support in the form of Junior Re-search Fellow.

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 6, MAY 2011 DOI 10.1002/jps