targeting and retention of magnetic targeted carriers (mtcs) enhancing intra-arterial chemotherapy

*Corresponding author. Fax: #310-206-2710.E-mail address: [email protected] (S. Goodwin)

Journal of Magnetism and Magnetic Materials 194 (1999) 132—139

Targeting and retention of magnetic targeted carriers (MTCs)enhancing intra-arterial chemotherapy

Scott Goodwin!,*, Caryn Peterson", Carl Hoh!, Craig Bittner!

!Department of Radiology, University of California Los Angeles Medical Center, Room BL-423 CHS, Los Angeles, CA 90095-1721, USA"FeRx Incorporated, San Diego, CA 90095-1721, USA

Abstract

MTCs were magnetically targeted and retained at a region of interest in a swine model after intra-arterial infusion.MTCs did not redistribute after removal of the magnetic field. Histopathology results demonstrated high particle densityin the area of the magnetic field. Particles were observed in the interstitium and occasionally intra-arterially. Regionaldelivery of chemotherapeutic drugs will be tested with this technology. ( 1999 Elsevier Science B.V. All rights reserved.

Keywords: Regional drug delivery; Magnetic targeting; Microparticle; Chemotherapeutic drugs

1. Introduction

The treatment of solid tumors using chemother-apy has been limited by systemic toxicity resultingin sub-optimal dosing, and by multiple other mech-anisms (e.g. multiple drug resistance of the tumorcells, tumor architecture limiting access of drug tothe tumor cells, volume of distribution for drug)resulting in limited efficacy. Regional therapyachieved through targeted drug delivery could im-prove efficacy by increasing the drug concentrationat the tumor while limiting systemic drug concen-trations. Higher drug concentrations at the tumormay be able to overcome the multiple drug-resis-tant phenotype, MDR1, by overcoming the P-gly-

coprotein pump that is functioning to pump drugout of cells. One type of regional therapy, magneticparticles carrying drug, may also achieve wide dis-persion throughout the tumor through the actionof the magnetic force on the particles. Regionaldrug delivery, however, will not be effective at treat-ing distant sites of tumor metastases unless thedrug is targeted to each known site. If systemicconcentrations of drug remain low after regionaldelivery, it may be possible to systemically adminis-ter chemotherapy coincident with regional therapyin order to optimize efficacy.

The use of magnetic targeted carriers (MTCs) fordrug delivery aims to target drug to specific sitesthrough the selective application of a magneticfield, and to achieve prolonged release of high,localized concentrations of drug by retention ofMTCs in the region of interest. Cells comprisingtumors are a non-synchronous population. Since

0304-8853/99/$ — see front matter ( 1999 Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 8 8 5 3 ( 9 8 ) 0 0 5 8 4 - 8

Fig. 1. Scanning electron micrographs of MTC particles. SEM shown in panel (a) is a 1000] magnification at an operating voltage of15 kV. The particles appear to be quite uniform in size and as shown in panel (b) are in the size range expected (0.5—5 lm).

chemotherapeutic drugs are active during specificphases of the cell cycle (e.g. DNA synthesis), a shortburst of drug may not kill a substantial portion oftumor cells. Retention of MTCs within the tumormay act as a drug reservoir. As drug desorbs fromthe particles over time, effective concentrations ofdrug may be maintained as more tumor cells cyclethrough S phase.

The use of magnetic carriers for drug delivery ofchemotherapeutic agents has evolved since the1970s when Widder et al. [1] developed albuminmicrospheres encasing the chemotherapeutic agentadriamycin, and using magnetite as the magneti-cally susceptible component. In their research, Dr.Widder and his associates first demonstrated inanimals the potential therapeutic benefit of mag-netically directing microspheres, containing adsor-bed drugs, into the capillary beds of tumors.Further development was not pursued due to theinadequate magnetic susceptibility of microsphereswhich restricted the application of the technologyto surface tumors. In the mid-1990s, Devineni et al.[2] compared the tissue distribution of methot-rexate following intra-arterial administration asa solution or as a magnetic microsphere conjugateto the brain. The authors concluded that once themagnetic field is turned off, the microsphere conju-gates exit the brain and redistribute to other sys-

temic organs, particularly lung. More recently,Lubbe et al. [3,4] used ferrofluids combined withepidoxorubicin in preclinical and early clinicalwork. These studies showed promise but theauthors concluded that larger magnetic particleswith higher magnetic susceptibility might be betterfocused and retained at the tumor site. In each ofthese studies, drug delivery was limited by the abil-ity to target and retain the majority of particles tothe region of interest.

The studies reported in this paper form the initialpart of the basis for a preclinical rationale formagnetic targeted delivery using MTCs. Imagingstudies and subsequent histopathology were com-pleted to test regional targeting and retention ofMTCs in a preclinical model. The swine model waschosen because (1) the size of the animal allowstesting of the magnetic targeting to depths of ap-proximately 13 cm, and (2) the architecture of thearterial vasculature of the liver, one of the targetedregions, is very similar to the human liver. Tocomplete a preclinical rationale for drug deliveryvia MTCs, it is also important to define the bindingand release of drug from the particles. Doxorubicinadsorbed to MTCs (MTC-DOX) has been initiallyselected for clinical development, and characteriza-tion of doxorubicin binding and release will bereported later.

S. Goodwin et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 132—139 133

Fig. 2. Whole-body gamma camera images following 99.Tc-MTC administration to a swine with magnetic targeting at t"60 min (a),and t"120 min (b) after the start of the infusion. Uptake of 99.Tc in thyroid, salivary glands and bladder is due to 15% unboundpertechnetate.

2. Composition and characterization of magnetictargeted carriers

MTCs are formed in a high-energy milling pro-cess in which activated carbon is incorporated into

metallic iron powder to produce a microparticlecomposite [5] with a 75 : 25, Fe : C ratio. The pro-cess has been optimized to provide a composite freeof iron oxides (Fe

2O

3) or cementite (Fe

3C alloy) as

measured by Mossbauer spectroscopy and X-ray

134 S. Goodwin et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 132—139

Table 1Analysis of radiographic images of the entire animal following 99.Tc-MTC administration at t"60 and 120 min from the start of theinfusion. 99.Tc-MTC administered intra-arterially through a segmental branch of the hepatic artery in the presence of a magnetic field

Organ Percent of total activity t"60 min Percent of total activity t"120 min

Bladder 4.0 4.2Salivary 1 10.9 10.9Salivary 2 10.3 11.2Thyroid NM! 3.4Cardiac pool 8.2 7.1Whole liver 66.6 63.1

Targeted liver 41.7 36.9

!NM"not measured.

diffraction. The resulting particles range in sizefrom 0.5 lm to approximately 5 lm, with 95% ofthe population distribution less than 3 lm (Fig. 1).Particles are subsequently gamma irradiated to 26kGy for sterility.

3. Labeling and administration of MTCs

To monitor short-term retention, MTCs werelabeled with the gamma-emitting isotope Techne-tium 99 (99.Tc) and then imaged using a gammacamera. 200 mg of MTCs were resuspended in10 ml of sterile saline. Approximately, 10 mCi of99.Tc was added to the vial. Particles were pelletedusing a magnet and the supernatant and pellet werecounted. The pellet (8.5 mCi) was resuspended in200 ml of sterile diluent to a final concentration of1 mg/ml (10% mannitol, 0.5% sodium car-boxymethylcellulose in 22 mM sodium phosphatebuffer at pH 6.5).

Prior to dose administration, a permanent neo-dymium magnet was positioned outside the bodyso that the magnetic field was directed to a pre-selected site in the liver or the lung. The magneticfield strength at the site ranged from 250 to 1000 G.MTCs were infused at 2 ml/min intra-arterially andallowed to flow towards the desired site and thusinto the magnetic field. Following administrationof MTCs, the magnet was held in place for anadditional 15—30 min. Angiograms were done im-mediately before the procedure to verify the place-ment of the catheter and to position the magnetproximal to catheter position. Following the infu-

sion, a post-dose angiogram was done to assess thepatency of the arteries. Images were taken witha gamma camera up to 3 h after administration toassess distribution of the 99.Tc activity.

To monitor long-term retention, unlabeledMTCs were administered as described above, andthe animal was imaged by MRI to follow the ironcomponent of the MTCs. MRI was performed onthe abdominal region of the animal including thestomach, liver, and bowel following administration.A follow-up MRI was repeated six days after dos-ing in order to determine whether particle distribu-tion was retained over this time period.

4. MTC targeting and retention

Whole-body gamma camera images (Fig. 2) weregenerated following administration of 99.Tc-MTCto the swine in the presence of a magnetic field asdescribed above. The first series of images (a) weretaken of the whole animal approximately 60 minafter administration, and a second series of images(b) were taken at approximately 2 h after MTCadministration. The images shown are those withdetectable signal, and Table 1 tabulates the distri-bution of activity across these sites. The resultsindicate that the radiolabel distributes to the tar-geted organ, liver, as well as to the salivary glands,thyroid, and bladder. Approximately 42% of thetotal activity measured was found in a very localiz-ed region of the liver, and almost 67% of the totalactivity is found in the targeted lobe of the liver.Non-targeted sites containing radiolabel are those


Fig. 3. Gamma camera images of the abdominal region of the swine after administration of 99.Tc-MTC. In panel (a) 50 mg 99.Tc-MTCwere infused in the absence of a magnet. At t"60 min after the start of the infusion, activity is dispersed throughout the entire liver (bothleft and right lobes) as well as in the cardiac pool (circled). In panels (b) and (c), 99.Tc-MTC particles were infused in the presence of anexternal magnet. In panel (b) at t"30 min after the start of the infusion, activity is localized to the magnetically targeted site within theright lobe of the liver. In panel (c), two separate administrations of 25 mg of 99.Tc-MTC were targeted to two locations within the samelobe of the liver. Immediately after completion of the second administration (t"30 min after the start of the first infusion), activity isseparately localized to two targeted regions within the left lobe of the liver.

Fig. 4. Gamma camera images of the chest region of the swine after administration of 50 mg of 99.Tc-MTC. 60 min after the start of theinfusion, activity is localized to the targeted lung and is absent from the liver (circled).

where free 99.Tc would be expected to distribute.Since MTCs were not washed after labeling anyresidual unbound 99.Tc in the form of pertech-netate would be taken up by the thyroid and thesalivary glands and eliminated in the urine. In sub-

sequent experiments, MRI imaging was unable todetect the presence of iron in these organs confirm-ing that the radiographic signal was not due to99.Tc-MTC. In images taken approximately75 min after removal of the magnet, the majority of


Fig. 5. MRI image of an abdominal section of the swine following administration of unlabeled MTCs under magnetic guidance to theright lobe of a swine liver. The MRI image shows the iron component of the MTCs circled in panel (a) on day 1 taken immediatelyfollowing infusion of the MTCs, and panel (b) shows the retention of the MTC particles 6 days following the infusion.

the activity still remains in the liver. The tissuedistribution achieved during the initial targeting ofMTCs did not change significantly for 2 h after theprocedure even though the magnetic field was nolonger present.

Regional distribution of MTCs is dependent onactive targeting by the magnetic field. Gammaradiographic images of the liver (Fig. 3) were gener-ated from three separate swine experiments inwhich 99.Tc-MTC was administered without mag-netic targeting or with magnetic targeting as de-scribed above. In the first animal (a), no magnetwas used and particles were distributed throughoutthe entire liver as a result of the normal process ofhepatic uptake of systemically administered par-ticles. In the second animal (b), a magnet was usedto localize or target the particles to a selected lobeof the liver. In the third animal (c), particles weretargeted in two separate administrations to twoseparate locations within the left lobe of the liver byrepositioning of the magnet and the catheter so thatthe magnetic field targeted different sites for eachadministration. The depths of MTC targeting inthese animals ranged from 8 to 12 cm. When MTCs

were administered to the liver and targeted witha magnet, 99.Tc was focused in the targeted por-tion of the liver with a signal density six-fold that ofany other site. By changing the position of themagnet and repeating a second administration ofparticles, two distinct sites in the same lobe of theliver showed radioactivity. After administration ofMTCs without a magnet, signal density in the liveris broadly distributed over the entire organ and isapproximately 40% that observed with magnetictargeting.

Targeting of MTCs to another organ, the lung,was achieved after intra-arterial infusion into theleft pulmonary artery. Whole-body radiographicimages were generated following administration of99.Tc-MTC to the swine in the presence of amagnetic field as described above. Free 99.Tc,representing 43% of the total counts, was againvisible in regions corresponding to the salivaryglands, thyroid, and bladder. The remaining activ-ity was localized in the left lung (Fig. 4) withalmost 50% of the activity measured in the lunglocalized to the site of magnet placement. The lackof radiographic signal in the liver (indicated by


Fig. 6. Photomicrographs of tissue samples stained with hematoxylin and eosin from the targeted lobe of the swine liver. Panel (a) showsa section from the targeted lobe of the liver viewed at 100] magnification, and panel (b) shows an enlargement at 400] magnification ofthe same section. The arrows show the location of the MTC particles.

Fig. 7. Photomicrographs of tissue samples stained with hematoxylin and eosin from the targeted left lung of the swine. Panel (a) showsa section from the targeted lung viewed at 100] magnification, and panel (b) shows an enlargement at 400] magnification of the samesection. The black spots indicate the presence of MTCs.

the circle in Fig. 4) implies that targeting ofMTCs occurs during the first pass. If MTCs werenot retained at the region of interest and sub-sequently entered the circulation, radiographicsignal due to 99.Tc-MTC would be expected in

the liver (refer to Fig. 3a) due to the normal processof hepatic uptake of systemically administeredparticles.

In subsequent targeting experiments, MTCs wereshown to be localized and retained for a period of six


days (Fig. 5) to the targeted lobe of the liver withoutsignificant redistribution as detected by MRI.

5. Histological analysis

At the conclusion of the imaging/retention stud-ies in liver, tissues were collected from targetedand non-targeted organs of the animals. The tissuesamples were stored in formaldehyde and submit-ted for histologic processing and microscopicevaluation. The presence of iron particles was read-ily visible in both the hematoxylin and eosin (H& E) and Prussian Blue stained sections. Aqualitative assessment of the microscopic analysisestimated about a ten-fold greater frequency ofMTC was seen in the targeted sections of liver ascompared to non-targeted sections (Fig. 6).The majority of the Fe particles were found withinportal triads or in periportal areas. In the portaltriads, MTCs were found in the wall of the hepaticartery, its branches, or within cells in connectivetissue around the hepatic artery. The Fe particleswere not present in clusters in the non-targetedliver lobe, and there was no evidence of accumula-tion in the arterial walls. The presence of MTCs inthe tissue outside of the vessels of the targeted lobeindicates that extravasation is a mechanism forretention of MTCs after removal of the magneticfield.

Following the imaging/retention studies in thelung targeting experiment, tissues were collectedfrom both lungs (targeted and non-targeted) as wellas heart, spleen and liver. The tissue samples werestored in formaldehyde and submitted for his-tologic processing and microscopic evaluation.Qualitative assessment of the microscopic analysisshow a ten-fold increase of MTCs in the targetedleft lung over the non-targeted right lung (Fig. 7),with no MTC deposition in heart, spleen, liver, ornon-targeted lung. MTC particles in the targetedlung lobe were seen consistently within cells in thealveolar septae with the preponderance of particlesbeing 1—2 lm in diameter. There was no evidence ofmorphologic damage to the lung tissues.

6. Summary

These experiments show that MTCs can be accu-rately targeted in a relevant animal model to regionsof interest in the liver and lung, with sufficient specifi-city to allow multiple administrations to different siteswithin the same region of interest. In the liver, MTCsare retained for a period of at least six days withoutsignificant redistribution as detected by MRI. Similarstudies in lung would be expected to demonstratesimilar retention. Histologic analyses confirm theenhancement of MTC distribution in the regions ofthe lung or liver chosen for magnetic targeting. Thepresence of MTCs in the tissue outside of the capil-laries indicates that extravasation of particles isa mechanism for retention of MTCs after removal ofthe magnetic field. These studies illustrate the ration-ale for using MTCs to which drugs are adsorbed totarget specific disease sites within the body. Theability to regionally target drug delivery predictsthat a reduced amount of drug can be given withthe intent of increasing the local concentration atthe desired site, therefore enhancing the efficacy ofthe treatment while decreasing systemic side effects.

The swine model has provided a clear rationaleof the use of MTCs for targeted delivery of pharma-ceutical agents. To demonstrate proof-of-principlefor use of MTCs as a targeted drug delivery system,the chemotherapeutic agent doxorubicin has beenchosen to investigate the safety and efficacy ofMTC-doxorubicin (MTC-DOX) in patients withhepatocellular carcinoma.

References

[1] K.J. Widder, R.M. Morris, G. Poore et al., Proc. Natl. Acad.Sci. 78 (1981) 579.

[2] D. Devineni, A. Klein-Szanto, J. Gallo, J. Neuro-Oncology24 (1995) 143.

[3] A.S. Lubbe, C. Bergemann, W. Huhnt et al., Cancer Res. 56(1996) 4694.

[4] A.S. Lubbe, C. Bergemann, H. Riess et al., Cancer Res. 56(1996) 4686.

[5] L.M. Allen, T. Kent, C. Wolfe et al., in: U. Hafeli, et al.,(Eds.), Scientific and Clinical Application of Magnetic Car-riers, Plenum Press, New York, 1997, p. 481.


targeting and retention of magnetic targeted carriers (mtcs) enhancing intra-arterial chemotherapy

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