Biodegradable nanoparticles — From sustained release formulations to improved site specific drug delivery

journal of

ELSEVIER

Journal of Controlled Release 39 (1996) 339-350

controlled release

Biodegradable nanoparticles From sustained release formulations to improved site specific drug delivery Jean-Christophe Leroux, Eric All6mann, Fanny De Jaeghere, Eric Doelker, Robert Gurny * School of Pharmacy, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland

Received 21 June 1995; accepted 28 September 1995

Abstract Biodegradable poly(DL-lactic acid) nanoparticles (NP) produced by the salting-out process were evaluated in vitro and in vivo for their sustained release properties and their capacity to temporarily avoid the mononuclear phagocyte system (MPS). In vivo, NP loaded with neuroleptic compound savoxepine were able to provide sustained plasma levels after intramuscular and intravenous injection. Intramuscularly injected NP remained at the site of injection, whereas intravenously injected NP were located mostly in the MPS. The NP were successfully coated during the preparation procedure with PEG 6000 and PEG 20 000. In vitro, these coatings provided a protective barrier against extensive uptake by human monocytes, at least in plasma. Analysis of plasma proteins adsorbed on NP and in vitro experiments on isolated cells revealed some differences between the opsonization process of plain and coated NP. Plain and PEG 20000-coated NP loaded with photosensitizer hexafluoro zinc phthalocyanine were injected intravenously to mice bearing EMT-6 mouse mammary tumors. The protective coating produced a dramatic increase of the photosensitizer concentration in blood and tumor as well as a decrease in the MPS sequestration. These findings suggest that surface-modified NP should prove useful for the delivery of chemotherapeutic drugs to tumoral tissues. Keywords: Nanoparticle; Poly(lactic acid); Opsonization; Sustained release; Drug targeting; Photodymanic therapy

1. Introduction Nanoparticles (NP) were introduced in the seventies as a possible means to improve site specific drug delivery (for review see Ref. [1]). At that time,

* Corresponding author.

liposomal formulations were still lacking stability and suffered from a relatively poor drug loading capacity. NP made of well characterized biodegradable polymers such as poly(alkyl cyanoacrylate) or poly(lactic acid) could be easily manufactured in a reproducible manner and represented an attractive alternative for improving the modulation of drug delivery, shelf life and stability in biological fluids [2]. However, initial expectations, for instance in cancer chemotherapy [3], were dampened by the fact

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that, following intravenous administration, NP were rapidly cleared from the systemic circulation. They ended almost exlusively in the mononuclear phagocyte system (MPS), mainly the macrophages of the liver and spleen. This extensive uptake of NP can be of clinical interest to overcome cancerous or infectious diseases affecting for instance the liver, because it can provide high local concentrations of chemotherapeutic agents [4-6]. However, the most usual purpose is to deliver NP to other sites than the MPS. The short half-life of NP is often associated with blood levels comparable to those of the free drug and with relatively poor drug concentrations in most tissues [7]. On the contrary, the use of NP remains a valuable therapeutic approach to obtain sustained or increased drug levels when appropriate administration modes are selected. For example, it was demonstrated that the precorneal residence time of pilocarpine was increased after incorporation of the drug into NP [8]. These applications, although interesting, are quite similar to those of microparticulate dosage forms. The use in the mid-eighties of amphiphilic coating agents capable of modifying the NP surface characteristics such that their clearance by the MPS be delayed or reduced, has opened new perspectives for improved drug-targeting [9]. Some poloxamines or poloxamers proved to decrease the accumulation rate of non biodegradable polystyrene and poly(alkyl methacrylate) NP in the liver and increase the deposition of the particles in other organs such as the kidneys [10]. Nevertheless, these coating agents often failed to provide a strong and stable steric barrier on biodegradable NP [11,12]. In 1987, Allen et al. [13] showed that it was possible to decrease the liposome uptake by incorporating in their structure specific glycolipids such as ganglioside GMt. This approach was based on mimicking the outer composition of red blood cells in order to produce liposomes that would not be recognized by the MPS. More recently, amphiphilic PEG such as PEG conjugated to phosphatidylethalonamine [14] were also used to protect liposomes from a rapid elimination. This resulted in higher plasma levels and increased accumulation of the cattier for instance, in solid tumors as compared to non sterically stabilized liposomes [15]. Accordingly, PEG or poly(ethylene oxide) chains were also included in the structure of

polymeric biodegradable NP. Preliminary in vitro and in vivo experiments have already shown the effectiveness of such coatings [16-18]. However, as yet little is known on how these coating agents interfere with the opsonization process and prevent the interaction of the NP with the cells of the MPS. Several investigations are presently underway in vitro and in vivo, in order to define a more rational approach for the development of sterically stabilized particles. The present paper deals with the use of biocompatible poly(lactic acid) NP prepared by the recently developed salting-out process, for sustained release dosage forms and improved site specific drug delivery [19]. This process does not require the use of chlorinated solvents and the particles thus prepared can be easily and rapidly purified by cross-flow filtration [20]. The particles are loaded with the lipophilic compound savoxepine (antipsychotic) and the kinetics are monitored in rats following intravenous (i.v.) and intramuscular (i.m.) injection. The influence of surface adsorbed PEG chains on the opsonization and uptake of the NP by human monocytes is evaluated and discussed. Finally, the biodistribution and pharmacokinetics of hexafluoro zinc phthalocyanine (ZnPcFl6) (photosensitizer for cancer chemotherapy) incorporated into plain and PEG 20000-coated NP in tumor bearing mice following i.v. injection of the particles is presented.

2. Materials and methods 2. I. Chemicals and polymers

Poly(DL-Iactic acid) (PLA) (Medisorb ~ 100DL) was supplied by Medisorb Technologies International L.P. (Cincinnati, OH). Poly(vinyl alcohol) (PVAL) was chosen as stabilizing colloid (Mowiol ® 4-88; Hoechst, Frankfurt/M, Germany). PEG 20 000 and 6000 and fluorescent tracer Nile Red were purchased from Sigma (Buchs, Switzerland). Savoxepine base (CGP 19486), [ 14C]savoxepine base (0.645 M B q / m g ) and [HC]savoxepine methanesulfonate (4.24 M B q / m g ) were provided by Ciba-Geigy. ZnPcFi6 (molecular mass 865 Da) was synthetised and purified as described previously [18]. The sol-

J.-C. Leroux et al./Journal of Controlled Release 39 (1996) 339-350

vents were of analytical grade and all other chemicals were commercially available.

2.2. Preparation of nanoparticles The NP were prepared by a salting-out procedure [19], as follows: an aqueous gel containing 35% ( m / m ) of magnesium acetate tetrahydrate and a variable amount of PVAL depending on the size desired for the NP, was added under vigorous stirring to an acetone solution containing the active compound or the fluorescent tracer and 5-18% ( m / m ) PLA, leading to the formation of a water-inoil emulsion. Despite the miscibility of water with acetone, a liquid-liquid two-phase system was formed due to the presence of the salting-out agent (magnesium acetate). Upon further addition of the aqueous gel, an oil-in-water emulsion was obtained. Finally, pure water was added, sufficient to allow diffusion of acetone into the aqueous phase with the result of forming spherical NP. The nanoparticulate suspension was purified by cross-flow filtration using a Sartocon ® Mini device (Sartorius, Grttingen, Germany) mounted with a polyolefin cartridge filter with a 100 nm pore size [20]. The NP were finally frozen at - 55°C, freeze-dried (0.05 mbar) and stored at - 25°C until use. The drug or tracer loadings were determined spectrophotometrically as described previously [ 17,18,21 ]. PEG-coated NP were prepared as described above, except that pure water was replaced in the last step by a 5 - 1 0 % ( m / m ) PEG 6000 or PEG 20000 aqueous solution. The PEG content of NP was assayed by colorimetry, as described earlier [17]. The size of the NP was assessed by photon correlation spectroscopy with a Coulter Nano-sizer ® (Coulter Electronics, Harpenden, Hertfordshire, UK).

2.3. In c,itro release kinetics A sufficient amount of NP (corresponding to 3 mg of savoxepine base) was suspended in a 0.15 M phosphate buffer (pH 7.4) containing 40 retool/1 of sodium lauryl sulfate (sink conditions). The suspension was agitated in a horizontal shaker maintained at 37°C. At selected time intervals, 2.5 ml of the suspension were sampled and centrifuged at 55 000 X g for 2 h. The absorbance of the supernatant was measured with a diode array spectrophotometer in the integrated wavelength range from 278 to 290 nm.

341

2.4. Uptake of fluorescent NP by human monocytes Fresh buffy coat was collected from healthy human donors and diluted 1:2 with Hanks' balanced salt solution. Mononuclear leukocytes were isolated according to the Leucosep TM protocol (Esquire Chemie AG, Ziirich, Switzerland). Cell viability was assessed by the trypan blue exlusion test and was found to be generally above 90%. The cell suspension was centrifuged at 400 X g for 10 min and the cell aggregate was resuspended in phosphate-buffered saline (PBS), human citrated plasma or human serum. Aliquots of 0.1 ml were sampled (1.2 × 106 cells) and incubated with 10 /xl of a 0.1% ( m / m ) Nile red-loaded NP suspension at 37°C for 30 min. At the end of the incubation time, cells were washed twice by centrifugation (400 × g) with 1 ml PBS and the cell-associated NP content was determined by flow cytometry using a FACScan (Beckton Dickinson, San Jose, CA) (reading channel: 559-611 nm). On the basis of light scatter properties only the signals associated with monocytes were analyzed [17].

2.5. Plasma protein adsorption pattern Nanopatticles (50 mg) were suspended in 1 ml of water. Aliquots (0.130 ml) were sampled and added to 1.3 ml of EDTA(K 3) anti-coagulated plasma collected from healthy human donors. The NP suspension in plasma was incubated 30 min at 37°C. Then, I ml of the suspension was collected, diluted 1:2.5 ( v / v ) with water and centrifuged at 11 000 X g for 5 rain. The supernatant was discarded and the pellet was resuspended with a denaturing solution containing 5.88% ( m / v ) sodium dodecyl sulfate and 1.36% ( m / v ) 1,4-dithioerythritol and incubated with this solution for 2 h at room temperature. The suspension was then centrifuged at 11 000 X g for 20 min and the supernatant was submitted to two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) as described elsewhere [17].

2.6. Animal studies 2.6.1. Administration of saxoxepine The experiments were approved by the ethical committee of the Canton of Basle, Switzerland. NP (736 nm) containing 8.36% ( m / m ) [14C]savoxepine

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J.-C. Leroux et al./ Journal of Controlled Release 39 (1996) 339-350

base were resuspended in saline to obtain a dispersion containing 10% ( m / v ) solids. NP were tested against a solution of [14C]savoxepine methanesulfonate at 5 m g / m l in saline. Healthy random bred pedigree male albino rats Tif:RAIf (SPF) weighing about 180-280 g were used. All animals had free access to water and food. The compound (expressed as savoxepine base) was either injected into the tail vein at 6.4 and 2.1 m m o l / k g or administered i.m. into the hind limb at 19.2 and 6.4 m m o l / k g for the nanoparticulate and solution formulations, respectively. Three rats were used per time point, and at the allotted times retroorbital blood was collected into heparinized tubes from animals under anesthesia. For the distribution study, three rats were killed after 24 h, and three rats after 7 days. Radioactivity in blood, plasma and tissues was measured by scintillation counting as described previously [22].

2.6.2. Administration of ZnPcFI6 All experiments were performed on male B A L B / c mice (19-23 g). These experiments were conducted following a protocol approved by the Canadian Council on Animal Care. All animals had free access to water and food. EMT-6 mouse mammary tumor cells were maintained as previously described [23]. A tumor was implanted on each hind thigh by intradermal injection of 2 × 105 EMT-6 cells suspended in 0.05 ml Waymouth growth medium (Gibco, Canada). Mice were used 10-11 days after cell inoculation when the tumor diameter and thickness reached 4 - 8 and 2 - 4 mm, respectively. Tumor bearing mice were injected i.v. via the caudal vein (0.2 ml) with 0.865 m g / k g ZnPcF16 formulated either in plain NP (464 nm, 0.66% m / m drug loading) or PEG 20 000-coated NP (988 nm, 0.61% m / m drug loading). Five mice were used per time point, and at the allotted times blood was collected from the axillary vessels in the angle of the forelimb by means of heparinized sytinges. Mice were killed after each time point and organs and tissues of interest were removed, washed with saline and blotted dry. Blood samples, tumors and aliquots of other organs were homogenized with DMF, incubated overnight at 37°C under mechanical agitation and then centrifuged as described elsewhere [18]. The ZnPcF16 concentration in the supernatant was assayed by fluorescence (Aex 766 nm, Aem 780 nm) [18].

3. Results and discussion

3.1. Extended release properties of nanoparticles Extended release properties of PLA colloidal carriers were first demonstrated in 1981 with testosterone-loaded NP prepared by the emulsion-evaporation method [24]. In the present work, assessment of such characteristics for particles prepared by the salting-out procedure is described. This new process has the advantage of avoiding the use of toxic solvents such as methylene chloride or chloroform. PLA was chosen as polymer for all of the studies because of its recognized biodegradability and lack of toxicity. Moreover, it is suitable for injection purposes and is used in commercialy available drugs formulated as microparticles (e.g. Parlodel ® LA). The salting-out procedure allowed the preparation of savoxepine loaded nanospheres. With this neuroleptic compound drug loadings reached up to 16.7%. Entrapment of the substance in the NP as high as 95% was observed, with an overall weight yield of the procedure after purification reaching 92%. These two points are of great relevance with regard to industrial applications. Moreover, largescale batches can be prepared easily with this technique. In vitro release studies demonstrated that this type of drag carriers allows an extended delivery of the drug over more than 1 week. Release patterns can be adjusted by varying several manufacturing parameters [21]. For example, NP of approximately 300 nm containing 16.7% of savoxepine released 90% of their content in less than 24 h, whereas particles of the same size containing 7.1% of the active compound released their content over a period of more than 3 weeks. The mean size of the NP represented also a major factor as shown in Fig. 1. NP of 303 and 671 nm released 50% of their content in 2.7 and 18.1 days, respectively. With larger particles, the 'burst' phase was noticeably reduced. However, it should be mentioned that y-sterilization of the freeze-dried particles accelerated the release profile. For example, for NP of 274 nm containing 7.1% of savoxepine, 50% of the drug was released in 1.4 days after a 25 kGy radiation (2.5 days before sterilization). This is apparently due to a dramatic decrease in number average molecular weight of PLA.

J.-C. Leroux et al. / Journal of Controlled Release 39 (1996) 339-350

The extended release characteristics of this NP formulation were then assessed in rats with [14C]savoxepine [22]. Cumulative elimination of radioactivity in feces and urine was recorded during 7 days. With NP of 736 nm, excretion of 50% of the injected dose of 14C took 3.4 and 5.4 days after i.v. and i.m. administration, respectively. As a comparison, for the savoxepine in solution, less than 1 day was necessary to eliminate the same amount of radioactivity. The kinetic study in plasma showed that following i.v. injection of free drug to rats (Fig. 2A), the concentration-time profile of 14C substances was biphasic. After a short distribution phase, the following elimination displayed a terminal h a l f - l i f e (tl/2) of 1.8 + 0.2 days. After i.m. injection of the solution (Fig. 2B), the very rapid absorption from the muscle to the blood compartment could not be observed. The plasma concentrations were almost superimposed with the i.v. curve 01/2 2.6 _+ 1.1 days). For NP, the 14C level decreased very rapidly after i.v. injection (Fig. 2A). This was attributed to the MPS uptake and was confirmed with high radioactivity levels found in the liver and spleen (55.2% and 8.4% of the injected dose after 24 h, respectively). After this very rapid distribution phase, the blood levels decreased very slowly (tl/2 of 20.7 _+ 8.5 days). With the i.m. injection of NP, high plasma levels of 14C substances were avoided. Absorption of the released compound at the injection site into the vascu-

A

0.1

0.01

I

1

40

i

I

3

'

I

4

i

I

5

i

I

i

6

7

B 1"

0.1"

0.01 1

2

3

4

5

6

7

Fig. 2. 14C plasma concentrations after i.v. (A) and i.m. (B) injection of a solution of [lac]savoxepine methanesulfonate ( 0 ) and of [ 14 C]savoxepine-loaded PLA NP ((3); mean + SD (n = 3). Concentrations were normalized to a dose of 6 m m o l / k g savoxepine base. Adapted from Ref. [22] with permission.

8O

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Time (d)

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60

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0

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343

20 0 0

5

10

15

20

25

30

Time (d) Fig. 1. Influence of the NP size on the release rate of savoxepine from PLA NP ( 0 , 303 nm; (3, 671 nm); m e a n + S D ( n = 4 ) . Adapted from Ref. [21] with permission.

lar compartment was sustained from the first to the seventh day. The plasma concentration maximum was obtained at 2 days. From this time point to 7 days, the plasma profile exhibited an exponential decay with a tl/2 of 5.4 ___2.2 days. From a kinetic standpoint (Fig. 2B), the i.m. injection of NP appeared to be the best way to provide sustained release of drug over a period of 7 days, since plasma levels of 14C varied only within a short range (0.04

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J.-C Leroux et al. / Journal of Controlled Release 39 (1996) 339 350 120 oo

100

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80

0

60

40 20

0

~ PBS

Plasma

Fig. 3. Relative uptake (cell-associated fluorescence) of plain ( • ) and PEG 6000-coated PLA NP ( [] ) by human monocytes, in PBS and plasma; mean + SD (n = 3).

to 0.02 nmol/g). Moreover, with this type of administration, most of the radioactivity remained at the site of injection and after 24 h only very low amounts of J4C were found in the liver (1.21% of the injected dose) and spleen (0.01%). Poly(alkyl cyanoacrylate) [25] and nanocapsules [26] allow also an extended delivery of drugs. However, due to the rapid degradation of this type of carriers, the release of drugs cannot generally be prolonged over more than 24 h.

3.2. Nanoparticles for improved site specific drug deliveo' In vitro uptake experiments of surface-modified NP were performed with human monocytes. These cells belong to the MPS and can be easily analyzed by flow cytometry. The influence of the PEG 6000 coating on the uptake of NP by human monocytes is shown in Fig. 3. These NP have an average size of 295 nm and contain only residual amounts of PEG 6000 (below 1.5%). Residual PEG 6000 still confer to PLA NP a protective barrier against extensive phagocytosis by human monocytes in plasma. In the absence of plasma proteins (PBS), the uptake is much lower and no difference between plain and PEG 6000-coated NP can be demonstrated. This may be explained by the ability of poly(ethylene oxide) chains to hinder the adsorption of plasma opsonins on the surface of NP. With PEG 20 000 a similar effect was noted in plasma but this coating also reduced the uptake of the particles in PBS [17]. This

may be explained by the higher content of PEG 20000 (16.2%) in NP and by the increase of the chain length. Indeed, it has been previously demonstrated in vitro and in vivo for NP that an increase of surface density and of the molecular weight of PEG coating is associated with a decrease of the particles interaction with the MPS [27-29]. In the case of liposomes, the molecular weight of PEG linked to distearoylphosphatidylethanolamine (DSPE) affects similarly the binding of liposomes to macrophages in vitro [30]. However, it seems that maximum circulation times in vivo can be achieved in the range of PEG 1000-DSPE to PEG 2000-DSPE, and further increase in the molecular weight of PEG has little effect on the half-life of liposomes [30]. The mechanism of action of the PEG coating is still unclear. It has been suggested that the highly hydrated poly(ethylene oxide) chains extend into a brush conformation, providing a steric stabilization of colloidal carriers. Such stabilized colloidal carriers are less prone to interact with cell membranes and opsonins [31]. Recent studies carried out especially with liposomes, have also shown that hydrophilicity alone is not sufficient and that the flexibility of the coating chains has to be sufficient to create a sort of 'dense cloud of probable conformations' [32,33]. The importance of the incubation medium for the screening of suitable coating agents appears to be of prime importance (Fig. 4). Indeed, in serum the protection offered by the steric stabilizer is reduced. In plasma, the addition of the anticoagulant may

120

100

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Fig. 4. Relative uptake (cell-associated fluorescence) of plain ( • ) and PEG 6000-coated PLA NP ([3) by human monocytes, in PBS, serum and heated serum; mean_+SD (n = 3).

345

J.-C. Leroux et al. / Journal of Controlled Release 39 (1996) 339-350

i n h i b i t the a c t i v a t i o n o f the c o m p l e m e n t s y s t e m b y c h e l a t i n g d i v a l e n t i o n s s u c h as C a 2÷, a n d thus m a s k

bile o p s o n i n s and can b e i n a c t i v a t e d after h e a t i n g the s e r u m at 56°C f o r 30 m i n [34]. A s s h o w n in Fig. 5,

the role o f c o m p l e m e n t in the p h a g o c y t o s i s o f the particles. T h e c o m p l e m e n t c o m p o n e n t s are h e a t la-

the h e a t i n a c t i v a t i o n o f the s e r u m p r o d u c e s a m a r k e d d e c r e a s e o f the u p t a k e o f plain a n d P E G 6 0 0 0 - c o a t e d

Table 1 Proteins possibly involved in the opsonization of nanoparticles NP

Size (nm)

Coating

Incubation medium

Incubation time (rain)

Results

PPA PASt a PS

900 1400 1200

-

Mouse serum

30

Poloxamers 235, 238 and 407 Poloxamine 908

Citrated human plasma(0.3-50%) Human serum (0.3-50%)

120

PS

60

Poloxamine 908

Rat serum fractions

n.m. b

PS

570

-

5

PS

60

Poloxamine 908

Citrated human plasma Citrated human plasma

Implication of complement components Artursson [37] in the opsonization of PASt NP Reduction in the total amount of pro- Norman [38] teins adsorbed on coated NP vs uncoated NP in plasma 0.3% and 50% and serum 0.3% IgG, complement C3, transferrin, fibronectin and fibrinogen (plasma 50%) found in similar quantities on coated and uncoated NP Involvement of heat stable proteins Moghimi [39] ( > 100 kDa and < 30 kDa) having dysopsonic properties Detection of 1000 protein spots Blunk [40]

PS

200

Poloxamer 238

Citrated human plasma

5

5

Poloxamine 908

PS PS

2001000 60

PS

1000

Poloxamer 407 Poloxamine 908

Human plasma

0.5-240

PLA

166 199 428

P E t chains Poloxamer 188 PEG 20000

Human serum 50% EDTA(K 3)-anticoagulated human plasma

1-60

PLA

-

Human plasma

n.m. b

Poloxamers 184, 188 and 407

Citrated human plasma

5

PASt, polyacrylstarch. b n.m., not mentioned.

a

30

Detection of 1200 and 820 protein spots for uncoated and coated NP, respectively Predominant proteins adsorbed on poloxamer 238-coated NP: fibrinogen and protein PLS: 6. Reduced adsorption of PLS: 6 and no adsorption of fibrinogen on poloxamine 908-coated NP Predominant proteins: fibrinogen and PLS: 6 Larger amount of proteins adsorbed on the more hydrophobic NP. IgG, transferfin, fibrinogen, albumin, apt CIII, apoJ and apt A1 less adsorbed on poloxamer 407-coated NP. Apt AIV and apt E less adsorbed on poloxamer 188-coated NP Reduction of the adsorption of PLS: 6 and of the proportion of adsorbed apt CIII with time. Opposite effect with apt E Marked decrease in complement consumption for PEt-coated NP Predominant proteins: albumin, IgG, IgM, antithrombin III and fibrinogen. Apt E and apt AIV found on uncoated but not on PEG-coated NP

Author (Ref.)

Blunk [41]

Blunk [42]

Blunk [43] Blunk [36]

Blunk [44]

Labarre [45] Leroux [17]

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J.-C, Leroux et a l . / Journal of Controlled Release 39 (1996) 339-350

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48

72

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96

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144

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168

Time(h) Fig. 5. ZnPcFl6 blood concentrationsafter i.v. injection of 0.865 mg/kg in EMT-6 tumour-bearingmice (O, plain PLA NP; ©, PEG 20000-coated PLA NP); mean, SE smaller than symbols (n = 5). No fitting for plain PLA NP because blood concentrations close to quantificationlimit.

NP. Accordingly, one can conclude that heat labile opsonins such as complement components, are probably involved in the uptake of PLA NP. Furthermore, in heat-treated serum, the difference between the uptake of plain and PEG-coated NP becomes again clearly discernible. At a first sight, serum would appear to be a more appropriate incubation medium since it contains no anticoagulant. However,

the analysis by 2-D PAGE of proteins which adsorbed onto PLA and other type of NP has shown that for instance fibrinogen, a typical plasma protein, is abundantly found on the surface of NP (Table 1). The adsorption of typical plasma proteins may interfere in vivo with the deposition of active complement components on the surface of NP and thus not correlate the in vitro findings. For instance, Dunn et al. [29] have shown that the addition of 5% ( v / v ) rat serum to the incubation medium, significantly reduced the difference between the uptake of uncoated vs PEG-2000-coated PS NP by non-parenchymal liver cells. However, the same authors were able to demonstrate that this PEG coating could increase dramatically the NP blood circulation time in vivo. A similar observation was made by our group with PEG 20 000-coated PLA NP. In serum, a reduction of the coating efficiency was also observed (data not shown). However, as it will be discussed below, these NP were shown to remain significantly longer in the systemic circulation as compared to plain particles. Accordingly, the use of plasma as incubation medium for the in vitro evaluation of steric stabilizers seems to be a reasonable compromise. One of the first approaches to evaluate the degree of opsonization of uncoated and coated NP was to measure the zeta potential of the particles after incubation in serum [35]. Generally, the adsorption of serum proteins onto the NP surface leads to an

Table 2 In vivo studies with coated PLA or PLGA nanoparticles NP

Adsorbed coating

Animal

Comments

Author (Ref.)

PLA

Human serum albumin

Rat

Bazile [ 4 6 ]

PLA

PEG 20000

Mouse

PLA-PEG 2000

-

Rat

Slight increase of AUC vs control NP Significant increase of AUC vs control NP Significantincrease of AUC for PEG-coated NP vs control NP

PLA PLGA

Poloxamer 188 PLA-PEG 2000 PLA-PEG 5000 Poloxamine 908

Significantincrease of AUC vs control NP

Stolnik [16]

PLGA-PEG 5000 PLGA-PEG 12000 PLGA-PEG 20000

Rat

Mouse

PLA-PEG < poloxamine 908 Significantincrease of AUC vs control NP. Blood circulation time of coated-NP increases with increasingmolecularweights of PEG

All6mann[18] Verrechia [47]

Gref [28]

J.-C Leroux et al. / Journal of Controlled Release 39 (1996) 339-350

increase of the absolute zeta potential value. This method can be used for the rapid screening of various coating agents but gives no information on the type of proteins involved in the opsonization process. Our group [17] and especially Blunk et al. [36], have shown that 2-D PAGE could be a useful tool for establishing a mapping of proteins adsorbed on NP. As illustrated in Table 2, the adsorption of plasma/serum opsonins on the NP surface appears to be a very complex process. Several proteins such as specific apolipoproteins like apolipoprotein E (apo E) were found to adsorb differently on plain vs coated NP [17]. However, it is yet not known if these proteins are of prime importance for the recognition process of foreign particles by the MPS. Some currently ongoing experiments tend to relativize the role of apolipoproteins in the uptake of PLA NP by human monocytes. In fact, as already demonstrated for liposomes [34], it appears that opsonins such as IgG and complement components play a major role in the opsonization process (to be published). Furthermore, any comparison between different experiments is difficult because the experimental conditions (e.g. incubation time, nature of carrier) influence the adsorption of proteins. Here again, the use of plasma or serum as incubation medium may significantly affect the results and more investigations are required to define precisely the optimum incubation conditions. Up to now, only few in vivo studies have been carried out with coated PLA or poly(DL-lactic-coglycolic acid) (PLGA) NP (Table 2). The steric stabilizer can be covalently bound to the polymer, mixed to it during the NP preparation or subsequently adsorbed to plain NP. We have chosen to coat PLA NP by adding PEG during the preparation

Table 3 Elimination half-life (tl/2) and AUC0_L68 h values of ZnPcFl6 incorporated into plain and PEG 20000-coated NP (from Ref.

[181) Formulation

Plain NP PEG 20 000-coated NP

t]/2

AUCo 168 h

(h)

(/xghg

10 30

57 227

J)

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1

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I

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12

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Time(h) Fig. 6. ZnPcFI6 tumour concentrations after i.v. injection of 0.865 m g / k g in EMT-6 tumour-bearing mice ( i , plain PLA NP; [], PEG 20000-coated PLA NP); mean _+SE (n = 5).

of NP, because the covalent attachment of PEG to PLGA or PLA, although certainly more stable, creates a new chemical entity. Poloxamine 908 and PEG of relatively high molecular weights significatively increase the area under the blood or plasma concentration-time curve (AUC) of biodegradable NP (Table 2). We demonstrated (Fig. 5 and Table 3) that the PEG 20 000 coating previously evaluated in vitro, could produce a dramatic increase of the blood circulation time of ZnPcF16 incorporated into PLA NP as compared to plain particles. The PEG content of these coated NP represented only 2% of the total NP mass [18]. After 24 and 168 h, the cumulated uptake of the compound in the liver and spleen represented 61% and 44% for plain NP vs 50 and 29% for PEG 20 000-coated NP, respectively. Generally, the amount of ZnPcFl6 found in the spleen was comprised between 10 and 20% of the administered dose. As stated elsewhere [48,49], this uptake could be reduced if the coated NP were smaller. The reduction of the uptake of the NP by the MPS and the resulting longer blood circulation time was associated with a higher deposition of the drug in the tumor (Fig. 6). This increase of drug concentration into the tumor results only from a passive targeting of the coated-NP, possibly because of the higher vascular permeability of many tumoral tissues [50]. Some photodynamic therapy experiments which are

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currently undertaken tend to d e m o n s t r a t e the benefit o f such coated carriers.

4. Conclusion Poly(DL-lactic acid) nanoparticles p r o d u c e d by the salting-out process allow an e x t e n d e d release o f drug o v e r variable periods o f up to 7 days, d e p e n d i n g on the size, drug loading and route of administration. A f t e r i.m. administration, these particles remain essentially at the injection site, and no significant a c c u m u l a t i o n is o b s e r v e d in the M P S . Therefore, this type of carrier appears to be a p r o m i s i n g alternative for the preparation o f sustained release formulations o f potent drugs with short half-lives, h a v i n g a m o l e c ular structure unsuitable for c h e m i c a l m o d i f i c a t i o n s such as grafting l o n g chain fatty acids. For site specific drug delivery, it is n o w generally admitted that surface m o d i f i c a t i o n s o f N P is a prerequisite to avoid temporarily the M P S . The present w o r k has s h o w n that such a m o d i f i c a t i o n with P E G can substantially p r o l o n g the circulation time o f nanoparticles and therefore, by passive targeting, increase the drug concentration in intradermal located tumors. To understand m o r e precisely the m e c h a n i s m s i n v o l v e d in the recognition o f N P by the M P S , important in vitro studies h a v e been undertaken. In particular, 2-D P A G E and cell e x p e r i m e n t s clearly indicate w h i c h proteins adsorb onto NP, but further investigations need to be carried out to identify the opsonins playing a significant role in the elimination o f N P f r o m the bloodstream. P E G appears to be a v e r y p r o m i s i n g steric stabilizer for b i o d e g r a d a b l e NP, but it will be necessary to d e t e r m i n e the o p t i m u m d e g r e e of stabilization. Indeed, it has been shown in the case of liposomes, that an important steric stabilization could e f f e c t i v e l y p r o v i d e a p r o l o n g e d circulation time, but hinder partially the active targeting o f l i p o s o m e s carrying on their surface h o m i n g devices such as m o n o c l o n a l antibodies [51,52].

Acknowledgements This w o r k was supported by funds f r o m the M e d i cal R e s e a r c h C o u n c i l of Canada, the Swiss National S c i e n c e F o u n d a t i o n and C i b a - G e i g y .

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