Design and preparation of gadolinium-loaded ...

S.TP. PHARMA SCIENCES 10 (1) 39-49 2000

Design and preparation of gadolinium-loaded chitosan particles for cancer neutron capture therapy H. Tokumitsu*1, H. Ichikawa1, T.K. Saha\ Y. Fukumori1 and LH. Block2 'Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Arise 518, Ikawadani-cho, Nishi-ku, Kobe 651-2180, Japan 2 School of Pharmacy, Duquesne University, 437 Mellon Hall of Science, Pittsburgh, PA 15282, United States 'Correspondence

Gadolinium-loadedchitosan

particulate devices for the gadolinium

Des systemes particulaires de chitosan charges de. gadolinium pour le traitement du cancer par capture de neutrons sont decrits. Tout cross-linked chitosan microspheres (Gd-DTPA-CMSs) were prepared d'abord, des microspheres de chitosan reticule renferman! de Tacide fry a conventional method using glutaraldehyde. The increase in gadopenteiique out etc preparees par la methode conventionnelle glutaraldehyde applied contributed to a size reduction and to the mettant en (cuvre le glutaraldehyde. I. 'augmentation de la quantite de formation of a reservoir structure via preferential surface-hardening glutaraldehyde employe entraine une diminution detaille desparticules with glutaraldehyde, but competitively led to a decrease in the el la formation d'une structure de type reservoir par durcissement gadolinium content of Gd-DTPA-CMSs. The smallest mass median preferentiel de la surface, mats, en contrepartie. conduit a une diameter of Gd-DTPA-CMSs was 1.9 am and the Gd content 6. \%, diminution de la teneur en gadolinium dans les microspheres. Le plus equivalent to a Gd-DTPA content of 21.2%. Next, a novel emulsion faible diametre moyen des microspheres de chitosan reticule est de droplet-coalescence technique was developed in order to prepare an 1,9 /.un el la teneur en gadolinium est de 6,1 %, correspondantd 21.29c injectable gadolinium-loaded chitosan particulate system without d'acide gadopenteiique. Ensuite, une nouvelle technique par cross-linking agents. This method is based on neutralization ofw/o coalescence d'emulsion a etc devetoppee de facon a preparer un emulsion droplets containing chitosan and Gd-DTPA and subsequent systeme particulate de chitosan charge en gadolinium sans agent de precipitation of the chitosan-Gd- DTPA complex caused by coalescence reticulation. Cette methode est fondee sur la neutralisation de with w/o emulsion droplets containing individually prepared sodium gouttelertes d'emulsions e/h renfermant du chitosan et de Tacide hydroxide. The gadolinium-loaded chitosan micro- and nanoparticles gadopenteiique. puis sur la precipitation du complexe cliilosan/acide produced using this technique hardly released Gd-DTPA in an isotonic gadopenteiique provoquee par la coalescence avec les goutteleltes phosphate-buffered solution over 7 days despite the high water d'une emulsion e/h renfermant de t'hydroxyde de sodium prepare solubility of Gd-DTPA, thus suggesting a strong interaction between separement. Les micro et nanoparticules de chitosan chargees en chitosan and Gd-DTPA. The optimized process conditions facilitated gadolinium produites par cette technique liberent difficilement sur production of gadolinium-loaded chitosan nanoparticles with an 7 jours I 'acide gadopenteiique en milieu tampon phosphate isotonique extremely high Gd-DTPA content (45.3%) and a suitable size for IV en depit de la forte solubilite dans I 'eau de I 'acide gadopenteiique, ce injection (452 run). Gadolinium-loaded chitosan nanoparticles qui laisse penser a une interaction entre le chitosan et Tacide displayed prolonged retention in tumor tissue after intratumoral gadopenteiique. L'optimisation des conditions operatoirespermet la injection in vivo. Consequently, this led to intensified tumor-growth production de nanoparticules de chitosan chargees de gadolinium suppression i n vivo in the gadolinium neutron-capture therapy trial by avec una ties forte teneur en acide gadopenteiique (45,3%) et une intratumoral injection. taille pour Tinjection IV (452 nm). Les nanoparticules de chitosan chargees presentent un long temps de retention dans le tissu tumoral apres injection intratumorale in vivo. Par consequent, ceci conduit a une diminution de la croissance tumorale dans les essais in vivo de traitement du cancer par capture de neutrons apres injection intratumorale.

neutron-capture therapy ofcancer are described in this paper. Firstly,

Key words: Chitosan — Gadopenlelic acid — Microsphere — Microparticle — Nanoparticle — Neutron capture therapy — Gadolinium — Emulsion — Coalescence — Cross-linking agent.

Mots clefs: Chitosan — Acide gadopenteiique — Microspheres — Microparticules—Nanoparticules — Therapie par capture de neutrons — Gadolinium — Emulsions — ('oalescence — Agent de reticulation.

Neutron-capture therapy (NCT) is a cancer treatment that utilizes nuclear neutron-capture reaction (NCR) of radiationproducing elements administered in vivo by thermal neutron irradiation. In the present NCT, boron-10 is typically used as the radiation-producing element | 1 | ; alpha particles with a short range (about 10 urn) and high linear energy transfer are emitted by boron-10 NCR (B-NCT) [2]. B-NCT has achieved encouraging results using mercaptoundecahydro-closododecaboratedianion(|BpHl IS! I | : -.BSH) in patients suffering from glioma grades IIl-IV [3] and using boronopbenylalanine (BPA) in patients suffering with malignant melanoma [4].

Therapeutic effects have, however, been highly dependent on the specific accumulation properties inside the aforementioned tumor cells; consequently, their use as boron-10 carriers has been limited to the types of tumor listed above. O n t h e other h a n d , g a d o l i n i u m n e u t r o n - c a p t u r e therapy ( G d - N C T ) utilizes the following N C R of g a d o l i n i u m - 1 5 7 . nonr a d i o e l e m e n t , by thermal neutron irradiation [5J: , 5 7 Gd + n (| —> l5s Gd + y-rays + internal conversion e l e c t r o n s - > 15S Gd + y-rays + A u g e r electrons + characteristic X-rays. T h e g a m m a - r a y s and A u g e r electrons thus generated yield a tumor-killing effect [6, 7 ] . G a d o l i n i u m has the following theoretical and potential

39

S.T.P. PHARMA SCIENCES 10 (1) 39-49 2000

Design and preparation of gadolinium-loaded chitosan particles for cancer neutron therapy H. Tokumitsu, H. Ichikawa, T.K. Sana, Y. Fukumori and LH. Blo

I. MATERIALS AND INSTRUMENTS

advantages over boron-10. Firstly, the thermal neutron-capture cross-section of gadolinium-157 is 66 times larger than that of boron-10 [8]. Secondly, the gamma rays are long-range (> 100 urn) compared with the alpha particles, so that they can affect tumors extensively, even if Gd is present exlracellularly in tumor tissue [91. Thirdly. Auger electrons with a short-range and high linear energy transfer may lead to the local, intensive execution of DNA in neoplastic cells [10]. Fourthly, since gadolinium has been generally used as a contrast enhancer in magnetic resonance imaging (MR1) diagnoses, well-designed gadolinium-loading particulate systems or compounds may well integrate Gd-NCT with MR! diagnosis in the future. The therapeutic potential of Gd-NCT has been explored in recent years [11-18). Magnevist (gadopentetatc dimeglumine aqueous solution), an MR1 contrast agent, has been mostly used as a source of gadolinium in Gd-NCT studies. Given the lackof targeting ability, however, an adequate amount of gadolinium needed to achieve an efficient therapeutic index could not be delivered via the intravenous (IV) route. In addition, even direct, intratumoral (IT) injection did not triggerthe significantly prolonged retention of gadolinium in tumor tissues. Thus, a key factor in the success of the current Gd-NCT trial is the use of a device by means of which gadolinium can be delivered efficiently and retained inside tumor tissues and/or cells during thermal-neutron irradiation. This may extend NCT to other types of tumor. Based on this perspective, we have prepared the delaycd-release form of ethyl cellulose-coated microcapsules containing Magnevist for the preliminary GdNCT trial [191. The microcapsules proved to be significantly effective in terms of survival time in the murine Ehrlich ascites tumor model 1201. This result led us to develop a more elaborate gadolinium-loaded particulate system and to establish its potential application to Gd-NCT. Fine particulate systems such as emulsions, liposomes and nanoparticles have actively been studied as injectable drug delivery systems (DDS) to enhance therapeutic potency and reduce side-effects [21 -34|. From a clinical point of view, they have to be biodegradable and/or highly biocompatible. Moreover, a high drug content is desirable because, in many cases, actual drug-loading efficiency is often loo low to secure an effective dose at the target site. Chitosan, a polysaccharide, has been widely studied as a material for DDS [35-371 due to its bio-erodible, biocompatible, bioadhesive and bioaclive characteristics [38-46]. These distinctive properties led us to use chitosan as a promising material for the design and preparation of a series of gadoliniumloaded particulate systems. In the present study, the crosslinked chitosan spheres containing gadopentetic acid (GdDTPA) (Gd-DTPA-CMSs) were first prepared using the conventional cross-linking technique. Furthermore, the noncross-linked blank chitosan microspheres (b-uCSs).Gd-DTPAloaded microparticles (Gd-uCPs) and nanoparticles (GdnanoCPs) were prepared using the novel emulsion-droplet coalescence technique. This paper describes the details of their development and the results of our recent NCT trial using the particulate systems llius obtained.

Gadopentetic acid (Gd-DTPA) [figure I) containing natural gadolinium was chosen as a source of gadolinium for the following reasons: I) Gd-DTPA has substantial chelate stability which is constant across a wide range of pH values; 2) its meglumine salt has been widely used as an NMR diagnostic agent (Magnevist, Schering AG, Germany) and has proved to have an exceptionally low toxicity [47.48]; 3) the high-loading efficiency of gadolinium in particles is anticipated because of the potential ionic interaction between the amino groups of chitosan molecules and Gd-DTPA molecules having twovalent anionic charges. Four types of chitosan (Katokichi Bio Co., Ltd., Japan) including grade 10B (100% deacetylated; viscosity of 0.5% w/v chitosan/0.2 M acetic acid buffer (pH 4.0) solution at 20°C, 53 inPas ), 9B (91.4% deacetylated; viscosity. 240 mPa-s). 8B (84.9% deacetylated; viscosity. 150 mPa-s) and 7B (74.2% deacetylated; viscosity, 325 mPa-s) were used. Gd 3 + HOOCH2C,

.CH2COOH NCH2CH2NCH2CH2N

OOCH2C

CH2COO-

CH2COCr

Figure 1 - Structures of gadopentetic acid (Gd-DTPA) [58].

Particle size distribution was measured by dispersing in methanol (Gd-DTPA-CMSs) or water (b-u.CSs and Gd-uCPs) using a laser-scattering size analyzer, and in water via a dynamic light-scattering technique (Gd-nanoCPs). Particle morphology was assessed by means of scanning electron microscopy (SEM). Gadolinium was assayed using inductivelycoupled plasma atomic emission spectrography (ICP-AES) aftersampleincineration;gadoliniumcontcnt(%)wasexpressed in terms of dry weight. Gd release was studied at 37°C via a method based on a dynamic dialysis system with cellulose tubing for Gd-DTPA-CMSs and Gd-nanoCPs. and by filtering the suspension after incubation with vigorous shaking in the case of Gd-uCPs. The gadolinium released in the sample solutions withdrawn at predetermined time intervals was measured by ICP-AES after incineration.

II. CROSS-LINKED, GADOLINIUM-LOADED CHITOSAN MICROSPHERES Many types of chitosan particles have been prepared by cross-linking methods using glutaraldchyde [49-51]. This method offers some advantages such as the formation of rigid particles and thus well-controlled drug-release [52-54]. Crosslinked chitosan microspheres containing Gd-DTPA (Gd-DTPACMSs) were therefore prepared first of all [55].

1. Preparation The previously reported method [50/ was used with a slight modification. In short, Gd-DTPA and 103 mg of chitosan 10B

40

S.T.P. PHARMA SCIENCES 10(1)39-49 2000

Design and preparation of gadolinium-loaded chitosan particles for cancer neutron therapy H. Tokumitsu, H. Ichikawa, T.K. Sana. Y. Fukumori and L.H. Block

were dissolved in 4 ml of a 5% aqueous solution of acetic acid. This solution was added to 60 ml of toluene containing Span 80, an emulsifier, with vigorous stirring using a Physcotron (NS50, N1TI-ON, Japan; open type NS-10 shaft, 22000 rpm, I h)or a Clearmix (CLM-0.8S, M-Technique Co., Ltd., Japan; R2rotor, 0.2 mm screen, open system, I4000 rpm, 15 min) as a homogenizer to produce a w/o emulsion. Glutaraldehydesaturated toluene (GST) [561 |to which Span 80 was added at 6% (w/v)], was added to the w/o emulsion and stirred overnight with a magnetic stirrer at room temperature to provide the cross-linked Gd-DTPA-CMSs. The resultant suspension was centrifuged to collect the Gd-DTPA-CMSs. The Gd-DTPACMSs thus separated were washed twice in succession with toluene, methanol, distilled water and acetone by centrifuging and then dried on silica gel under vacuum conditions at room temperature.

•—i—->

1

2

4

1

i 6

i

i

•

8

• 10

Gd-DTPA/chitosan ratio (-)

2. Morphology, particle diameter and Gd-DTPA content Figure 2 illustrates changes in the mass median diameter and the gadolinium content of the Gd-DTPA-CMSs prepared by modifying the amount of Gd-DTPA (A) or Span 80 (B) applied. As shown in figure 2A, an increase in Gd-DTPA triggered a single decrease in the mass median diameter (D50) of Gd-DTPA-CMSs, nevertheless increasing above that of the Gd-DTPA-free CMSs above 1.7 of the Gd-DTPA/chitosan ratio indicated by the arrow. The SEM observation indicated that Gd-DTPA-CMSs partially contained nonspherical fragments and aggregates whereas the Gd-DTPA-free CMSs were entirely smooth-surfaced and spherical. In particular, GdDTPA-CMSs prepared at 9.7 of the Gd-DTPA/chitosan ratio were completely nonspherical, rough-surfaced, collapsed and/ or aggregate-shaped. The gadolinium content sharply increased to 12.5% (corresponding to 43.5% as Gd-DTPA) at 1.7 of the Gd-DTPA/chitosan ratio, thereafter saturated at about 13%. The high loading efficiency of Gd-DTPA in Gd-DTPACMSs would be associated with the possible electrostatic interaction between the amino groups in chitosan and the carboxylic groups in Gd-DTPA |49]. An increase in the amount of Gd-DTPA applied would trigger a rise in the number of amino groups of chitosan presumably interacting with GdDTPA in the aqueous droplets during emulsification. If the I: I ion-pair formation of the amino groups and the carboxylic groups occurred entirely in the absence of Span 80 and glutaraldehyde, the theoretical maximum content of gadolinium or Gd-DTPA would be about 18% or 63% respectively. This would clearly explain why the gadolinium content was saturated at 1.7 of the Gd-DTPA/chitosan ratio, corresponding to 63% of the Gd-DTPA content. The electrostatic interaction of chitosan with Gd-DTPA will always rival the cross-linking by glutaraldehyde during emulsification and hardening in the preparation process. Glutaraldehyde dissolved in the continuous medium of toluene would be widely available for cross-linking, mainly on the droplet surface [54,57). This may cause the gadolinium content to fall to about 13%. The presence of an excess amount of GdDTPA above 1.7 of the Gd-DTPA/chitosan ratio would prevent glutaraldehyde from cross-linking, leading to poor surface-

o

5

10

15

20

Weight of Span 80 in toluene (g) Figure 2 - Effects of the applied weight ratio of Gd-DTPA to chitosan (A) and the amount of Span 80 (B) on the size (O) and Gd content (•) of Gd-DTPA-CMSs [55].

hardening and, consequently, to size-enlargement of Gd-DTPACMSs by aggregation with the small amount of Span 80 (420 mg) applied. This shows that the Gd-DTPA/chitosan ratio is a very important factor in governing the size and shape of GdDTPA-CMSs as well as gadolinium content. Gd-DTPA-CMSs prepared at a 1.7 ratio of Gd-DTPA/chitosan will be referred to below as Gd-DTPA-CMS (L). An increase in the amount of Span 80 in toluene used for the primary emulsion at a Gd-DTPA/chitosan ratio of 1.7 {figure IB) caused a marked decrease in the mean particle size (D50) of the resulting Gd-DTPA-CMSs, whilst the gadolinium content did not decrease up to a level of 5 g of Span 80. Above 5 g of Span 80, however, the size was virtually unaltered accompanied by a fall in gadolinium content. The reduction in gadolinium content in Gd-DTPA-CMSs prepared with 20 g of Span 80 showed that excess Span 80 reduced the incorporation of GdDTPA in chitosan. The effect on size distribution and gadolinium content of the GST volume applied was then investigated. The results are shown in table /. As theGST volume increased, size distribution narrowed together with a decrease in D50 and gadolinium content. This suggests that the higher cross-linking density might competitively hinder the electrostatic interaction between chitosan and Gd-DTPA, probably close to the particle surface rather than inside Gd-DTPA-CMSs, which would account for the smaller diameter. Following application of 50 ml of GST volume, most of the Gd-DTPA-CMS (P50) presented with a 41


Design and preparation of gadolinium-loaded chitosan particles for cancer neutron therapy H. Tokumitsu. H. Ichikawa, T.K. Sana, Y. Fukumori and L.H. Block

smooth-faced and spherical shape on SEM examination. Moreover. Gd-DTPA-CMS (P50) had the sharpest size distribution of all the products prepared with a Physcotron (D5Q, 2.3 pm: gadolinium content, 5.8%, corresponding to 20.2% as Gd-DTPA). In contrast, the least cross-linked GdDTPA-CMS (PI) had a broad particle size distribution despite application of copious quantities of Span 80 (20 g). This suggested highly frequent aggregation due to excessively poor surface-hardening. Sufficient quantities of GST and Span 80 were obviously needed to produce the discrete spheres but this triggered a decrease in Gd content.

3. Release of Gd-DTPA The in vitro release of Gd-DTPA from three types of GdDTPA-CMSs, (L), (P50) and (C), was investigated in an isotonic phosphate buffer of pH 7.4. The results are shown in figure 4A. The Gd-DTPA-CMS (L) released 50% of the gadolinium loaded in the particles within 50 min (T50 =50 min). The release was, however, prolonged with Gd-DTPA-CMS (P50) and (C): T50 = 2.1 and 2.4 h respectively. Infigure4B. the linear relations between the percent released and the square root of time were obtained below 70% released after a 2-5 min lag time. Infigure4C, the relations between the logarithm of the percent remaining and time were more completely linear. Gd release kinetics seem to obey the first-order release mechanism rather than the square root of time equation (Higuchi equation). This suggests that the Gd-DTPA-CMSs might be a reservoir type as indicated from the experimental studies outlined above.

Table I - Effect of GST volume on CMS size and Gd content and comparison between Gd-DTPA-CMS(P) and Gd-DTPA-CMS(C), prepared by Physcotron and Clearmix, respectively' (55]. Gd-DTPA-CMS

P1 P10 P50 C

GST vol.2 (ml)

D10 (prn)

D50 < M m)

1 10 50 50

1.8 1.5 1.3 1.2

5.1 3.1 2.3 1.9

D903 Gd content (Mm) (%) 14.9 8.5 3.5 3.1

12.7 8.5 58 6.1

' The applied weight ratio of 1.7 between Gd-DTPA and chitosan in 4 ml of 5% acetic acid aqueous solution, and the volume of toluene (60 ml) containing 20 g of Span 80 were kept constant, respectively. 2 GST would contain 0.02 mol% glutaraldehyde; that is. about 1.9 mg of glutaraldehyde in 10 ml. For ail amino groups of 103 mg chitosan to be cross-linked, about 32 mg glutaraldehyde would be required. 3 D10, D50 and D90 are the particle sizes at 10, 50 and 90% of cumulative size distribution, respectively.

Instead of Physcotron, a different homogenizer (Clearmix) was used for primary emulsification in the same formulation as that of the Gd-DTPA-CMS (P50) with the smallest D50 and the sharpest particle size distribution. The size distribution and the gadolinium content of the Gd-DTPA-CMSs (Gd-DTPA-CMS (O) thus prepared are also shown in table I with illustration of the scanning electron micrograph infigure3. The Gd-DTPACMS (C) was similar to the Gd-DTPA-CMS (P50) in terms of D50, particle size distribution and gadolinium content but displayed a more spherical, discrete shape.

nQ)

3 = oe 20

Is °-4 *•"* w

15

if « (d •*

10

uo o >

50

c

Si

3.3. Generation mechanism of Gd-|iCPs The generation mechanism of Gd-uCPs appears to differ from that of the above b-uCSs. Gd-uCP generation might involve not only simple neutralization but also a strange precipitation process in the chitosan medium. In fact, whereas the droplet size of emulsion A was almost identical (about 4 urn), regardless of whether Gd-DTPA or acetic acid was used, droplet morphology (figure 9) and particle diameter differed significantly. Moreover, the mass median diameter of Gd-uCPs did not vary even with increasing Gd-DTPA (acid) concentrations in chitosan-dissolving droplets {figure 10). In addition, the quantity of Gd-DTPA incorporated was scarcely released from the Gd-uCPs for a long time. This suggests that Gd-uCPs might be produced through the highly stable complex formation of Gd-DTPA with chitosan. In the Gd-uCPs. chitosan precipitation from the medium containing Gd-DTPA would be

•D-° a 0

()

50

100

150

200

250

Gd concentration (ppm) measured by ICP Figure 11 - Gadolinium microanalysis on Gd-uCP suspension and dilute Magnevist solution using prompt y-ray emission by thermal neutron irradiation (58]. • , Gd-jjCP suspension ; O, dilute Magnevist solution.

4. Gd-DTPA-incorporated chitosan nanoparticles (Gd-nanoCPs) 4.1. Particle diameter and Gd-DTPA content Gd-nanoCPs were prepared by modifying the emulsiondroplet coalescence technique {figure 4} [59]. The mean particle

45


Design and preparation of gadolinium-loaded chitosan particles for cancer neutron therapy H. Tokumitsu, H. Ichikawa, T.K. Sana, Y. Fukumori and L.H. Blo

diameter and gadolinium content of Gd-nanoCPs prepared using chitosan 10B and 10% Gd-DTPA solution as chitosan medium by the emulsion-droplet coalescence technique were 426 nm and 9.3% respectively, equivalent to 32.4% as GdDTPA. SEM observation confirmed that the particle size of GdnanoCPs with chitosan 10B was virtually similar to that of the primary particles of agglomerated Gd-pCPs. This suggests that the present process, in which the continuous phase was changed only from chloroform to liquid paraffin, succeeded in preventing the nanoparticles from agglomeration. The effects of the Gd-DTPA concentration in an aqueous chitosan solution on the particle size and gadolinium content of Gd-nanoCPs are shown in table It. The particle size of GdnanoCPs with chitosan 10B was not significantly affected by the Gd-DTPA concentration in the chi tosan-dissolving droplets, which was the same as in the Gd-pCPs. However, gadolinium content increased up to 13.0% (corresponding to45.3% as GdDTPA) with an increase in Gd-DTPA concentration. The latter might trigger an increase in gadolinium content and, consequently, in particle volume by incorporating larger quantities of Gd-DTPA. Conversely, if two-valent anionic GdDTPAexhibitedacross-iinkingeffect by electrostatic interaction with cationic chitosan, the increase in Gd-DTPA concentration would reduce particle volume via a de-swelling effect. As a result, these rival effects might result in the unchanged particle size.

chitosan solvent in emulsion A droplets with sodium hydroxide in emulsion B droplets and subsequent or simultaneous ionic interaction of the amino groups of chitosan with Gd-DTPA as well as Gd-pCPs. However, the precipitation of Gd-nanoCPs might have occurred without further agglomeration within the emulsion droplets. In fact, mean diameter did not reflect the emulsion-droplet size whereas the mass median diameter of the previous Gd-pCPs (about 4 pm) corresponded to the droplet size of emulsion A. Then, Gd-DTPA was more enriched in GdnanoCPs as compared with previous Gd-pCPs: when 100% deacetylated chitosan was used, the gadolinium content was approximately 3-4 times higher than that of the corresponding Gd-pCPs. This high incorporation (45.3%) of Gd-DTPA in chitosan particles seems to be linked with the generation of discrete nanoparticles via a particularly fast precipitation process induced by a strong ionic-interaction. Interestingly enough, this happened only following replacement of the continuous medium with liquid paraffin instead of chloroform. Although the grounds for nanoparticle generation have not been fully elucidated, fortunately for us, this preparation technique using liquid paraffin produced Gd-nanoCPs with an extraordinarily high Gd-DTPA content (45.3%) and of a size suitable for IV injection (452 nm) [62], 4.3. In vitro Gd-DTPA release properties The in vitro Gd-DTPA release from Gd-nanoCPs prepared with chitosan 10B and 10% Gd-DTPA solution depended considerably on the types of release medium used [59]. In PBS, Gd-nanoCPs released only 1.8% of gadolinium (Gd-DTPA) over 7 days; this behavior pattern was similar to that of GdpCPs but different from that of the Gd-DTPA-CMSs. This once again suggests a strong complex formation of Gd-DTPA with chitosan in a simple aqueous medium. In contrast, 55% of gadolinium (Gd-DTPA) incorporated into the Gd-nanoCPs were eluted in human plasma for 3 h respectively. This fast release was not burst. Moreover, it might not be due to chitosan degradation because 100% deacetylated chitosan has proved not to be easily degradable [44]. The detailed release mechanism is still under investigation. This rapid release in plasma may not be conducive to the delivery of gadolinium to the tumor site via IV injection (or infusion) in Gd-NCT and NMR diagnosis. This is because free Gd-DTPA would not be distributed preferentially to the target tissues and eel Is via the IV route and rapidly el i minated with the urine, which is a well-known fact.

Table II - Mean particle size and Gd content of Gd-nanoCPs prepared with various deacetylated degrees of chitosan and Gd-DTPA concentrations in chitosan media [59]. No.ol batch

Mean part. Gd content (% w/w)* size (nm)* [Gd-DTPA content. %]

Chitosan 10B - 5 % Gd-DTPA sol.

3

461115

-10% Gd-DTPA sol.

6

426 ± 28

-15% Gd-DTPA sol.

3

452 ± 25

Chitosan 9B -10% Gd-DTPA sol.

3

594 ± 96

4.1 1 1.0 [14.213.41

Chitosan 8B -10% Gd-DTPA sol.

3

750177

3 . 3 1 0.8 [11.612.7)

7.71 1.7 [26.9 ± 5.9) 9.3 ± 3.2 [32.4111.01 13.011.8 [45.31 6.2]

The effects of the degree of chitosan deacetylation are also illustrated in table H. As the degree of chitosan deacetylation decreased, the mean particle diameters gradually increased accompanied by a decrease in gadolinium content. This suggests that the lower capacity for ion-pair formation at a lower degree of deacetylation would lead not only to less incorporation of gadolinium, resulting in a diminished de-swelling effect, but also to slower precipitation resulting in enhanced particle growth.

4.4. In vivo Gd-DTPA retention in tumor tissue In vivo retention of gadolinium (Gd-DTPA) in tumor tissue was investigated by direct subcutaneous IT injection of 200 pi of isotonic Gd-nanoCP suspension into male C57BL/6 mice with solid B16F10 malignant melanoma which grew to around 10 mm in diameter [59]. Dilute Magnevist solution was injected in the same way to elicit comparison. Each dosage form was administered at the rate of 1200 pg gadolinium. Following administration of the Gd-nanoCP suspension. 68.4% of the dose of gadolinium administered (820.6 pg) was present in the tumor block at 24 h post-injection. Contrastingly, only 0.4% of the dose of gadolinium administered (5.3 pg) remained in the

4.2. Generation mechanism of Gd-nanoCPs With this method, Gd-nanoCPs were generated by precipitation of chitosan triggered by neutralization of the 46

Design and preparation of gadolinium-loaded chitosan particles for cancer neutron therapy H. Tokumitsu, H. Ichikawa, T.K. Sana, Y. Fukumori and L.H. Block

S.T.P. PHARMA SCIENCES 10(1) 39-49 2000

tumor block 24 h after administration of a dilute solution of Magne vist. This meant that Gd-nanoCPs retained a large amount of Gd-TPA for a longer period in tumor tissue whilst Gd-DTPA was rapidly eliminated following administration as a solution. Rapid in-vitro release of Gd-DTPA of this nature from GdnanoCPs, as observed in human plasma, was not observed invivo in tumor tissue.

DTPA). An increase in the amount of glutaraldehyde and surfactant applied led to a size reduction and enhanced particle shape as well as to a decrease in the gadolinium content of GdDTPA-CMSs. The electrostatic interaction between chitosan and Gd-DTPA and preferential surface-hardening by glutaraldehyde contributed to the formation of fine, spherical, Gd-enriched and prolonged-release CMSs with a mass median diameter of 1.9 u.m, a Gd content of 6.1 % and a 50% dissolution time of 2.4 h. A novel emulsion droplet-coalescence method was developed in order to prepare finer, injectable particulate systems. This method involves precipitation of chitosan in an aqueous acidic Gd-DTPA solution by neutralization and subsequent ionic interaction of the amino groups of chitosan with Gd-DTPA produced by coalescence of the acidic and alkali droplets in the emulsion mixture. Basic studies of preparation conditions in b-uCSs without Gd-DTPA and GduCPs paved the way for the chitosan nanoparticulate system (Gd-nanoCPs) with a considerably highGd content, long-term suppressed release of Gd in a simple aqueous medium and a remarkably long retention time in the tumor after IT administration in vivo. In the Gd-NCT trial conducted in vivo, tumor growth in mice given Gd-nanoCP was significantly suppressed by neutron irradiation despite the smaller quantity of gadolinium administered compared with earlier in-vivo Gd-NCT studies. In this particular study, the novel gadolinium-incorporated chitosan nanoparticles (Gd-nanoCPs) proved to be extremely useful as an IT injectable gadolinium carrier for Gd-NCT implying that, even in NCT, the design and preparation of pharmaceutical dosage forms can be a promising way of achieving improved clinical efficiency.

IV. GD-NCT TRIAL USING GD-NANOCPS ,

'

'

'

A Gd-NCT trial was carried out in male C57BL/6 mice subcutaneously bearing solid, radio-resistant B16F10 malignant melanoma [631 using Gd-nanoCPs with chitosan 10B [64]. Two hundred |il of Gd-nanoCP suspension or dilute Magnevist solution were directly injected twice into the solid tumor tissue 24 and 8 h before neutron irradiation of a total dose of 2400 ug as natural gadolinium per mouse. Thermal neutron beams with an average fluorescence of 6.32 x 10 u neutrons/cm2 on the tumor surface were irradiated only once to caged mice according to Kurri [65,66]. Tumor growth was significantly suppressed with the Gd-nanoCPs administered and in the neutron-irradiated (Gd-P, N+) group despite the radio-resistant melanoma model employed, the prolonged interval (8 h) until neutron irradiation after the second gadolinium injection and administration of a smaller dose of gadolinium than that used in previous in-vivo Gd-NCT trials. The mean tumor volume at 14 d after neutron irradiation was less than 15% of that in the neutron-irradiated (Gd-, N+) group that did not receive gadolinium. Incontrast.no evidcnceof inhibited tumor growth entirely similartothat in the [Gd-. N+] group was apparent in the mice in the group receiving dimeglumine gadopentetate solution and neutron irradiation (Gd-S, N+) despite the fact that the same gadolinium dose level was administered as in the nanoparticle formulation. These results could be ascribed to the long interval between administration and neutron irradiation as Magnevist would be eliminated rapidly from tumor tissue after IT injection (the biological half-life of the wash-out period was about 20 min) [12]. The survival time in the [Gd-P, N+] group was also significantly prolonged. In particular, 3 out of 6 mice (50%) survived for more than 28 days after neutron irradiation, which was approximately more than twice the mean survival rate of mice in other groups. The marked suppression of tumor growth observed in the present Gd-NCT trial with the Gd-nanoCPs was clearly due to excellent Gd-DTPA retention properties in tumor tissue following IT injection. These results indicate that GdnanoCPsarea very useful gadolinium carrier for IT injection in solid tumors in Gd-NCT.

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The design and preparation of gadolinium-loaded chitosan particulate devices for Gd-NCT are described in this paper. The cross-linked Gd-DTPA-CMSs were prepared via the conventional cross-linking method using glutaraldchyde. Following application of Gd-DTPA at levels higher than the equimolar ratio of its carboxylic groups to chitosan amino groups, the gadolinium content of the Gd-DTPA-CMSs was saturated at about 13% (corresponding to about 45% as Gd-

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MANUSCRIPT Accepted for publication 10 December 1999.

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