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Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides
Journal of Controlled Release 53 (1998) 137–143
Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides Elias Fattal a , Christine Vauthier a , Isabelle Aynie a , Yuichiro Nakada a ,b , Gregory Lambert a , c a, Claude Malvy , Patrick Couvreur * a
Laboratoire de Physico–chimie–Pharmacotechnie–Biopharmacie, URA CNRS 1218, Universite´ Paris-Sud, Faculte´ de Pharmacie, 92296 Chatenay-Malabry, Cedex, France b Pharmaceuticals Research Centre, Kanebo, Ltd., 5 – 90, Tomobuchi-cho, 1 -Chome Miyakojimaku, Osaka 534, Japan c URA CNRS 147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France Received 7 May 1997; received in revised form 15 July 1997; accepted 16 July 1997
Abstract Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered. Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic / phagocytotic pathway. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting. Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles. These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g. liver metastasis or hepatitis). 1998 Elsevier Science B.V. Keywords: Polyalkylcyanoacrylate nanoparticles; Adsorption; Intracellular delivery; Tissue distribution
1. Introduction Antisense oligonucleotides with base sequences complementary to a specific RNA offer the exciting potential to selectively modulate the expression of an *Corresponding author. Tel.: 133 1 4683 5396; fax: 133 1 4661 9334.
individual gene [1,2]. However, crucial problems such as the poor stability of oligonucleotides versus nuclease activity in vitro and in vivo and the low intracellular penetration have to be solved [3,4]. Different chemical modifications have been performed in order to protect oligonucleotides and to improve their cellular uptake. However, in some cases, especially with phosphorothioates, non spe-
0168-3659 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0168-3659( 97 )00246-0
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E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
cific effects were observed [5]. Two techniques were also proposed to increase oligonucleotide penetration into cells: electropermeabilization [6] and microinjection [7], which both are supposed to introduce oligonucleotides directly into cell cytoplasm, avoiding lysosomes. However, these methods cannot be applied in therapy for humans. Thus, the development of particulate carriers for oligonucleotide delivery may be considered as being more realistic to improve the in vivo efficacy of these molecules by protecting them against degradation and by increasing their intracellular delivery. We have recently developed an original method allowing the efficient association of oligonucleotides with biodegradable polyalkylcyanoacrylate (PACA) nanoparticles. This association was achieved by the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations such as quaternary ammonium salts [8]. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of nanoparticle capture by an endocytotic / phagocytotic pathway [9]. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice [10] and the stability of these molecules has been evaluated by an original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by a multichannel radioactivity counting [11]. Finally, as reported by Schwab et al. [12], when applied to anti-ras, this original delivery system markedly inhibited Ha-ras-dependent tumour growth in nude mice after intratumoural injection. The aim of this paper is thus to review the potential of polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides.
2. Materials and methods
IGR, Villejuif, France. The 59-end labelled oligonucleotides were synthesized using T4 polynucleotide kinase and 33 P-ATP (Amersham, France).
2.2. Nanoparticles preparation and adsorption of oligonucleotides Nanoparticles were prepared as previously described by Couvreur et al. [13] using an emulsion polymerization process. The monomers used were either isobutylcyanoacrylate (IBCA) or isohexylcyanoacrylate (IHCA). Oligonucleotide adsorption onto nanoparticles was achieved by adding oligomers to the polymeric suspension in the presence of cetyltrimethylammonium bromide (CTAB), as ionpairing agent [8]. Practically, CTAB (final concentration, 300 mM) and 33 P-labelled oligonucleotides (final concentration, 1 mM) were added to the nanoparticles suspension in the presence of 150 mM NaCl.
2.3. Oligonucleotide analysis Oligonucleotide stability in vitro and in vivo was measured by electrophoresis on a 20% polyacrylamide-7 M urea sequencing gel (PAGE). Bands were analyzed quantitatively by measuring the radioactivity using an automatic TLC-linear analyzer [11]. After electrophoresis, slab polyacrylamide gels were placed horizontally under the ionization detection chamber of a multi-channel radioactivity counter (M.C.R.C.) (Automatic TLC-Linear Analyzer, Berthold, Wildbad, Germany) as shown. The radioactivity distribution profile was derived from the recorded counts and analyzed under computer control. The decomposition of each electropherogram was done and these simple integrations were used to measure the area under each profile between any two boundaries. A background zone was chosen downstream of each peak and subtracted from the original gel image.
2.1. Materials 2.4. Cell culture experiments 59-phosphorylated oligothymidylate (pdT 16) was purchased from Pharmacia Biotech (St Quentin-enYvelines, France). The 15-mer oligonucleotide, with a sequence complementary to the AUG region of the env mRNA of Friend retrovirus was obtained from
For oligonucleotide uptake assays, U937 cells were incubated with 59-end labelled hexadecathymidylate oligonucleotide (5nM) free or associated with PIHCA nanoparticles (5 mg / ml) using the
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
139
conditions published previously [8]. Cell uptake was monitored as the ratio of radioactivity in cell lysate against total radioactivity (cell1medium).
2.5. Pharmacokinetic and in vivo studies Pharmacokinetic studies were performed using male OF1 mice receiving injections into the tail vein of 33 P-pdT 16 free or adsorbed onto nanoparticles at a dose of 5 nmol / 5 ml / kg corresponding to a dose of polymer of 5 mg / kg [10]. Blood and organs were removed for the determination of the radioactivity by scintillation counting after digestion of the tissues. In vivo stability of pdT 16 was also investigated after extraction of oligonucleotides from tissues followed by PAGE electrophoresis as described above.
Fig. 1. Schematic representation of oligonucleotides adsorption at the surface of polyalkylcyanoacrylate nanoparticles coated with a hydrophobic cation.
3. Results and discussion
3.1. Nanoparticle preparation and adsorption of oligonucleotides The association of oligonucleotides with PIBCA and PIHCA nanoparticles was found to occur only in the presence of a hydrophobic cation such as CTAB [8]. The poor yield of oligonucleotide association in the absence of hydrophobic cations may be explained by the fact that PACA nanoparticles bear negative charges which induce an electrostatic repulsion with the polyanionic oligonucleotides. In addition, the hydrophilic character of the nucleic acid chains which are known to be soluble in water does not allow the interaction with the polymer through hydrophobic bounds. In the proposed method, oligonucleotide adsorption on the nanoparticles was mediated by the formation of ions pairs between the negatively charged phosphate groups of the nucleic acid chain and the hydrophobic cations (Fig. 1). Fig. 2 shows the decrease of the zeta potential of the particles as a function of the oligonucleotide concentration. The maximum loading capacity may be considered as being between 5000 and 6000 oligonucleotides molecules per particle [14]. The adsorption efficiency of oligonucleotide-cation complexes on PACA nanoparticles was found to be highly dependent on several parameters: oligonucleotide chain length, nature of the cyanoacrylic polymer,
hydrophobicity of the cation used as ion-pairing agent and ionic concentration of the medium [8].
3.2. In vitro stability of oligonucleotides nanoparticles When adsorbed onto PIHCA nanoparticles, oligonucleotides were totally protected against enzymatic degradation even after five h incubation with phosphodiesterase [8]. In addition, we determined that about 90% of the oligonucleotide still remained intact after overnight incubation with the enzyme
Fig. 2. Zeta potential (d) and 15-mer oligothymidilate loading (s) of polyalkylcyanoacrylate nanoparticles.
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E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
(0.1 mg / ml). This result suggests that phosphodiester linkages or the 39-end of oligonucleotides were not accessible to enzymatic attack. We then compared the stability of oligonucleotides either free or adsorbed to nanoparticles in 70% of mouse plasma at 378C. The half-lives of pdT16 incubated free and bound to the nanoparticles were 6.0 and 12.5 min, respectively [10]. Thirty minutes after incubation in plasma, the percentage of unchanged 33 P-pdT 16 was 2.9% for free oligonucleotides whereas 28.9% of 33 P-pdT 16 associated with nanoparticles was still intact [10].
3.3. Cell uptake studies Cell uptake studies of 15-mer adsorbed onto nanoparticles was performed in non toxic conditions. It was observed that the uptake of oligonucleotide was dramatically increased when associated with nanoparticles (Fig. 3). After 24 h incubation, uptake of oligonucleotide was eight-times higher when adsorbed to nanoparticles [9]. The cellular uptake of oligonucleotide nanoparticles was markedly reduced (95%) at 48C as compared to 378C. These results show that oligonucleotides adsorbed onto nanoparticles were internalized in U937 cells by an endocytic / phagocytic process rather than simply adsorbed at the membrane surface. This has been confirmed by confocal microscopy with fluorescently labelled
Fig. 3. Cell (U 937) uptake of a 15-mer oligothymidilate free (s) or associated with polyisohexylcyanoacrylate nanoparticles (d) (24 h incubation, 378C); from [9].
nanoparticles. The polymeric structure of PIHCA nanoparticles exclude their cellular uptake by fusion. After internalization, nanoparticles accumulate into phagosomes or lysosomes and it may be supposed that when released in such intracellular compartments, oligonucleotides will be rapidly degraded. This is the reason why the intracellular stability of internally labelled 15-mer free or adsorbed to PIHCA nanoparticles has been tested at various incubation times [9]. No intact oligonucleotide was detected in cell lysate after 1.5 h incubation with free oligonucleotide. On the contrary, the 15-mer adsorbed onto PIHCA nanoparticles remained completely intact after 6.5 h of incubation. At 24 h, some degradation products appeared, but the fraction of intact oligonucleotides remained considerable. The intracellular distribution of oligonucleotides (59 labelled oligomer) was measured after lysis in presence of Nonidet-P40, a nonionic detergent which protects the nuclear structure. It has to be noted that the cytoplasmic fraction contains also endocellular vesicles like lysosomes or phagosomes. About 20% of the oligonucleotide given free or delivered by PIHCA nanoparticles were found in the nuclear fraction. Intact oligonucleotide was found in the nuclear but not in the cytoplasmic fraction after incubation (6 h) with the free oligonucleotide [9]. This could be explained by the presence in the cytoplasmic fraction of lysosomes which are expected to be the major site of oligonucleotide degradation. On the contrary, when adsorbed onto nanoparticles, intact oligonucleotides were detected in both nuclear and extranuclear fraction. The increased stability of the oligonucleotide adsorbed onto nanoparticles which is observed in the extranuclear fraction could be explained by the fact that nanoparticles protect them against digestion by lysosomal enzymes. How they escape from the lysosomes still remains unclear, but it is supposed that CTAB which is a quaternary ammonium could destabilize the lysosomal membrane, thus allowing the release of the oligonucleotide into the cell cytoplasm.
3.4. In vivo studies with oligonucleotide nanoparticles The pharmacokinetic studies [10] have shown that
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
141
although nanoparticles did not markedly increase the blood half-life of the 33 P-pdT 16, its tissue distribution was significantly modified. Indeed, when transported by PIBCA nanoparticles, 33 P-pdT 16 was importantly taken up by the liver whereas a subsequent reduced distribution in the other organs was observed, especially in the kidney (Fig. 4). These data suggest that with the aid of nanoparticles, the oligonucleotide could be delivered to the liver with a certain specificity and the urinary excretion decreased. To address the crucial problem of oligonucleotide degradation, we have investigated the state in which (degraded or not) oligonucleotide was in the plasma and delivered to the liver [10]. It should be pointed out that the evaluation of the in
vivo stability of oligonucleotides represents a major technical difficulty. Two techniques are generally utilized to quantify this degradation: autoradiography and HPLC. None of these methodologies are able to combine a high sensitivity with an accurate quantitative determination of the undegraded oligonucleotide. We have, thus, proposed an original method that consists in the use of a TLC analyser allowing, in polyacrylamide gels, to quantify the amount of undegraded pdT 16 in tissues such as liver and plasma [11]. This method is able to distinguish between oligonucleotides differing by only one nucleotide in length (Fig. 5). Using that methodology, it was found that a significant amount of 33 P-pdT 16 was kept intact in the liver (Fig. 6) and the plasma when administered under the form of nanoparticles [10]. Finally, this system has been proved to be efficient in vivo when applied to an
Fig. 4. Liver (A) and Kidney distribution of 33P-pdT 16 free (s) or associated to PIBCA nanoparticles (d) after IV administration in mice.
Fig. 5. Electrophoretic profile of 15-mer (a), 16-mer (b) and a mixture of both oligonucleotides (c) on a 24 cm high dried gel; from [11].
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E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
diseases such as viral hepatitis or liver cancers or metastasis.
Acknowledgements This paper has been partly supported by a grant of the CNRS (GDR n8 1207) and by the ARC (Association pour la Recherche contre le Cancer).
References
Fig. 6. Electrophoresis chromatogram of intact pdT 16 in liver 5 min after intravenous administration (5 nmol / kg) of 33P-pdT 16 associated to PIBCA nanoparticles (a) or of 33 pdT 16 free (b). (c) is the control (liver homogenate spiked with 18 000 dpm of intact 33 pdT 16); from [10].
anti-ras oligonucleotide: this system markedly inhibited Ha-ras-dependent tumour growth in nude mice in a highly specific manner [12].
4. Conclusion In conclusion, the results presented in this review show that the association of oligonucleotides with biodegradable PACA nanoparticles is possible if using a hydrophobic counter-ion. This system has been proved to be efficient not only for protecting the oligonucleotide from the degradation by 39-exonucleases but also for increasing the intracellular capture of these molecules. Organ distribution profile of oligonucleotides could be dramatically modified after association with PACA nanoparticles with an increase of liver concentration. These results suggest that nanoparticles might be considered as an interesting carrier system for the antisense treatment of liver
´` ´ Specific regulation of gene expres[1] C. Helene, J.J. Toulme, sion by antisense, sense and antigene nucleic acids, Biochem. Biophys. Acta 1049 (1990) 99–125. [2] G. Zon, Oligonucleotides as potential therapeutic agents, Pharm. Res. 5 (1988) 539–549. [3] S.L. Loke, C.A. Stein, X.H. Zhang, K. Mori, M. Nakanishi, C. Subasinghe, J.S. Cohen, L.M. Necker, Characterization of oligonucleotide transport into living cells, Proc. Natl. Acad. Sci. USA 86 (1989) 3474–3478. [4] L. A Yakubov, E.A. Deeva, V.F. Zarytova, E.M. Ivanova, A.S. Ryte, L.V. Yurchenko, V.V. Vlassov, Mechanism of oligonucleotide uptake by cells: Involvement of specific receptors, Proc. Natl. Acad. Sci. USA 86 (1989) 6454–6458. [5] C.A. Stein, Phosphorothioate antisense oligodeoxynucleotides: questions of specificity, Trends Biotechnol. 14 (1996) 147–149. [6] L.M. Mir, H. Banou, C. Paoletti, Introduction of definitive amounts of non permeant molecules into living cells after electropermeabilization: direct access to the cytosol, Exp. Cell. Res. 175 (1988) 15–25. [7] P. Dash, L. Lotan, M. Knapp, E.R. Kandel, P. Goelet, Selective elimination of mRNAs in vivo complementary oligonucleotide promote RNA degradation by an RnaseH like activity, Proc. Natl. Acad. Sci. USA 84 (1987) 7896– 7900. [8] C. Chavany, T. Le Doan, P. Couvreur, F. Puisieux, C. ´` Helene, Polyalkylcyanoacrylate nanoparticles as polymeric carries for antisense oligonucleotides, Pharm. Res. 9 (1992) 441–449. [9] C. Chavany, T. Saison-Behmoaras, T. Le Doan, F. Puisieux, ´` P. Couvreur, C. Helene, Adsorption of oligonucleotides onto polyisohexylcyanoacrylate anoparticles protects them against nucleases and increases their cellular uptake, Pharm. Res. 11 (1994) 1370–1378. [10] Y. Nakada, E. Fattal, M. Foulquier, P. Couvreur, Pharmacokinetics and biodistribution of oligonucleotide adsorbed onto polyisobutylcyanoacrylate nanoparticles after intravenous administration, Pharm. Res. 13 (1996) 38–43. [11] I. Aynie, C. Vauthier, M. Foulquier, C. Malvy, E. Fattal, P. Couvreur, Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143 counter for the evaluation of oligonucleotide-bound drug carrier, Anal. Biochem. 240 (1996) 202–209. [12] G. Schwab, C. Chavany, I. Duroux, G. Goubin, J. Lebeau, C. ´` Helene, T. Behmoaras, Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice, Proc. Natl. Acad. Sci. USA 91 (1994) 10460–10464.
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[13] P. Couvreur, M. Roland, P. Speiser, Process for preparing biodegradable submicroscopic particles containing a biologically active substance and their use, United States Patent (1984) n8 4 489 055 ´ [14] G. Lambert, E. Fattal, J. Feger, P. Couvreur, personnal communication (1996).
Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides Elias Fattal a , Christine Vauthier a , Isabelle Aynie a , Yuichiro Nakada a ,b , Gregory Lambert a , c a, Claude Malvy , Patrick Couvreur * a
Laboratoire de Physico–chimie–Pharmacotechnie–Biopharmacie, URA CNRS 1218, Universite´ Paris-Sud, Faculte´ de Pharmacie, 92296 Chatenay-Malabry, Cedex, France b Pharmaceuticals Research Centre, Kanebo, Ltd., 5 – 90, Tomobuchi-cho, 1 -Chome Miyakojimaku, Osaka 534, Japan c URA CNRS 147, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France Received 7 May 1997; received in revised form 15 July 1997; accepted 16 July 1997
Abstract Antisense oligonucleotides with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using oligonucleotides as active molecules, the development of polymeric particulate carriers was considered. Oligonucleotides were associated with biodegradable polyalkylcyanoacrylate nanoparticles through the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of the capture of nanoparticles by an endocytotic / phagocytotic pathway. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice and the stability of these molecules has been evaluated by original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by multichannel radioactivity counting. Stability in vivo in the plasma and in the liver was shown to be improved when the oligonucleotides were adsorbed onto the nanoparticles. These results obtained both in vitro and in vivo open exciting perspectives for the specific delivery of oligonucleotides to the liver, thus considering this approach for the treatment of liver diseases (e.g. liver metastasis or hepatitis). 1998 Elsevier Science B.V. Keywords: Polyalkylcyanoacrylate nanoparticles; Adsorption; Intracellular delivery; Tissue distribution
1. Introduction Antisense oligonucleotides with base sequences complementary to a specific RNA offer the exciting potential to selectively modulate the expression of an *Corresponding author. Tel.: 133 1 4683 5396; fax: 133 1 4661 9334.
individual gene [1,2]. However, crucial problems such as the poor stability of oligonucleotides versus nuclease activity in vitro and in vivo and the low intracellular penetration have to be solved [3,4]. Different chemical modifications have been performed in order to protect oligonucleotides and to improve their cellular uptake. However, in some cases, especially with phosphorothioates, non spe-
0168-3659 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0168-3659( 97 )00246-0
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E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
cific effects were observed [5]. Two techniques were also proposed to increase oligonucleotide penetration into cells: electropermeabilization [6] and microinjection [7], which both are supposed to introduce oligonucleotides directly into cell cytoplasm, avoiding lysosomes. However, these methods cannot be applied in therapy for humans. Thus, the development of particulate carriers for oligonucleotide delivery may be considered as being more realistic to improve the in vivo efficacy of these molecules by protecting them against degradation and by increasing their intracellular delivery. We have recently developed an original method allowing the efficient association of oligonucleotides with biodegradable polyalkylcyanoacrylate (PACA) nanoparticles. This association was achieved by the formation of ion pairs between the negatively charged oligonucleotides and hydrophobic cations such as quaternary ammonium salts [8]. Oligonucleotides bound to these nanoparticles were found to be protected from nuclease attack in cell culture media and their cellular uptake was increased as the result of nanoparticle capture by an endocytotic / phagocytotic pathway [9]. The in vivo pharmacokinetic profile of oligonucleotides free or associated with nanoparticles has been investigated after intravenous administration to mice [10] and the stability of these molecules has been evaluated by an original methodology based on the use of polyacrylamide gel electrophoresis (PAGE) followed by a multichannel radioactivity counting [11]. Finally, as reported by Schwab et al. [12], when applied to anti-ras, this original delivery system markedly inhibited Ha-ras-dependent tumour growth in nude mice after intratumoural injection. The aim of this paper is thus to review the potential of polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides.
2. Materials and methods
IGR, Villejuif, France. The 59-end labelled oligonucleotides were synthesized using T4 polynucleotide kinase and 33 P-ATP (Amersham, France).
2.2. Nanoparticles preparation and adsorption of oligonucleotides Nanoparticles were prepared as previously described by Couvreur et al. [13] using an emulsion polymerization process. The monomers used were either isobutylcyanoacrylate (IBCA) or isohexylcyanoacrylate (IHCA). Oligonucleotide adsorption onto nanoparticles was achieved by adding oligomers to the polymeric suspension in the presence of cetyltrimethylammonium bromide (CTAB), as ionpairing agent [8]. Practically, CTAB (final concentration, 300 mM) and 33 P-labelled oligonucleotides (final concentration, 1 mM) were added to the nanoparticles suspension in the presence of 150 mM NaCl.
2.3. Oligonucleotide analysis Oligonucleotide stability in vitro and in vivo was measured by electrophoresis on a 20% polyacrylamide-7 M urea sequencing gel (PAGE). Bands were analyzed quantitatively by measuring the radioactivity using an automatic TLC-linear analyzer [11]. After electrophoresis, slab polyacrylamide gels were placed horizontally under the ionization detection chamber of a multi-channel radioactivity counter (M.C.R.C.) (Automatic TLC-Linear Analyzer, Berthold, Wildbad, Germany) as shown. The radioactivity distribution profile was derived from the recorded counts and analyzed under computer control. The decomposition of each electropherogram was done and these simple integrations were used to measure the area under each profile between any two boundaries. A background zone was chosen downstream of each peak and subtracted from the original gel image.
2.1. Materials 2.4. Cell culture experiments 59-phosphorylated oligothymidylate (pdT 16) was purchased from Pharmacia Biotech (St Quentin-enYvelines, France). The 15-mer oligonucleotide, with a sequence complementary to the AUG region of the env mRNA of Friend retrovirus was obtained from
For oligonucleotide uptake assays, U937 cells were incubated with 59-end labelled hexadecathymidylate oligonucleotide (5nM) free or associated with PIHCA nanoparticles (5 mg / ml) using the
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
139
conditions published previously [8]. Cell uptake was monitored as the ratio of radioactivity in cell lysate against total radioactivity (cell1medium).
2.5. Pharmacokinetic and in vivo studies Pharmacokinetic studies were performed using male OF1 mice receiving injections into the tail vein of 33 P-pdT 16 free or adsorbed onto nanoparticles at a dose of 5 nmol / 5 ml / kg corresponding to a dose of polymer of 5 mg / kg [10]. Blood and organs were removed for the determination of the radioactivity by scintillation counting after digestion of the tissues. In vivo stability of pdT 16 was also investigated after extraction of oligonucleotides from tissues followed by PAGE electrophoresis as described above.
Fig. 1. Schematic representation of oligonucleotides adsorption at the surface of polyalkylcyanoacrylate nanoparticles coated with a hydrophobic cation.
3. Results and discussion
3.1. Nanoparticle preparation and adsorption of oligonucleotides The association of oligonucleotides with PIBCA and PIHCA nanoparticles was found to occur only in the presence of a hydrophobic cation such as CTAB [8]. The poor yield of oligonucleotide association in the absence of hydrophobic cations may be explained by the fact that PACA nanoparticles bear negative charges which induce an electrostatic repulsion with the polyanionic oligonucleotides. In addition, the hydrophilic character of the nucleic acid chains which are known to be soluble in water does not allow the interaction with the polymer through hydrophobic bounds. In the proposed method, oligonucleotide adsorption on the nanoparticles was mediated by the formation of ions pairs between the negatively charged phosphate groups of the nucleic acid chain and the hydrophobic cations (Fig. 1). Fig. 2 shows the decrease of the zeta potential of the particles as a function of the oligonucleotide concentration. The maximum loading capacity may be considered as being between 5000 and 6000 oligonucleotides molecules per particle [14]. The adsorption efficiency of oligonucleotide-cation complexes on PACA nanoparticles was found to be highly dependent on several parameters: oligonucleotide chain length, nature of the cyanoacrylic polymer,
hydrophobicity of the cation used as ion-pairing agent and ionic concentration of the medium [8].
3.2. In vitro stability of oligonucleotides nanoparticles When adsorbed onto PIHCA nanoparticles, oligonucleotides were totally protected against enzymatic degradation even after five h incubation with phosphodiesterase [8]. In addition, we determined that about 90% of the oligonucleotide still remained intact after overnight incubation with the enzyme
Fig. 2. Zeta potential (d) and 15-mer oligothymidilate loading (s) of polyalkylcyanoacrylate nanoparticles.
140
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
(0.1 mg / ml). This result suggests that phosphodiester linkages or the 39-end of oligonucleotides were not accessible to enzymatic attack. We then compared the stability of oligonucleotides either free or adsorbed to nanoparticles in 70% of mouse plasma at 378C. The half-lives of pdT16 incubated free and bound to the nanoparticles were 6.0 and 12.5 min, respectively [10]. Thirty minutes after incubation in plasma, the percentage of unchanged 33 P-pdT 16 was 2.9% for free oligonucleotides whereas 28.9% of 33 P-pdT 16 associated with nanoparticles was still intact [10].
3.3. Cell uptake studies Cell uptake studies of 15-mer adsorbed onto nanoparticles was performed in non toxic conditions. It was observed that the uptake of oligonucleotide was dramatically increased when associated with nanoparticles (Fig. 3). After 24 h incubation, uptake of oligonucleotide was eight-times higher when adsorbed to nanoparticles [9]. The cellular uptake of oligonucleotide nanoparticles was markedly reduced (95%) at 48C as compared to 378C. These results show that oligonucleotides adsorbed onto nanoparticles were internalized in U937 cells by an endocytic / phagocytic process rather than simply adsorbed at the membrane surface. This has been confirmed by confocal microscopy with fluorescently labelled
Fig. 3. Cell (U 937) uptake of a 15-mer oligothymidilate free (s) or associated with polyisohexylcyanoacrylate nanoparticles (d) (24 h incubation, 378C); from [9].
nanoparticles. The polymeric structure of PIHCA nanoparticles exclude their cellular uptake by fusion. After internalization, nanoparticles accumulate into phagosomes or lysosomes and it may be supposed that when released in such intracellular compartments, oligonucleotides will be rapidly degraded. This is the reason why the intracellular stability of internally labelled 15-mer free or adsorbed to PIHCA nanoparticles has been tested at various incubation times [9]. No intact oligonucleotide was detected in cell lysate after 1.5 h incubation with free oligonucleotide. On the contrary, the 15-mer adsorbed onto PIHCA nanoparticles remained completely intact after 6.5 h of incubation. At 24 h, some degradation products appeared, but the fraction of intact oligonucleotides remained considerable. The intracellular distribution of oligonucleotides (59 labelled oligomer) was measured after lysis in presence of Nonidet-P40, a nonionic detergent which protects the nuclear structure. It has to be noted that the cytoplasmic fraction contains also endocellular vesicles like lysosomes or phagosomes. About 20% of the oligonucleotide given free or delivered by PIHCA nanoparticles were found in the nuclear fraction. Intact oligonucleotide was found in the nuclear but not in the cytoplasmic fraction after incubation (6 h) with the free oligonucleotide [9]. This could be explained by the presence in the cytoplasmic fraction of lysosomes which are expected to be the major site of oligonucleotide degradation. On the contrary, when adsorbed onto nanoparticles, intact oligonucleotides were detected in both nuclear and extranuclear fraction. The increased stability of the oligonucleotide adsorbed onto nanoparticles which is observed in the extranuclear fraction could be explained by the fact that nanoparticles protect them against digestion by lysosomal enzymes. How they escape from the lysosomes still remains unclear, but it is supposed that CTAB which is a quaternary ammonium could destabilize the lysosomal membrane, thus allowing the release of the oligonucleotide into the cell cytoplasm.
3.4. In vivo studies with oligonucleotide nanoparticles The pharmacokinetic studies [10] have shown that
E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
141
although nanoparticles did not markedly increase the blood half-life of the 33 P-pdT 16, its tissue distribution was significantly modified. Indeed, when transported by PIBCA nanoparticles, 33 P-pdT 16 was importantly taken up by the liver whereas a subsequent reduced distribution in the other organs was observed, especially in the kidney (Fig. 4). These data suggest that with the aid of nanoparticles, the oligonucleotide could be delivered to the liver with a certain specificity and the urinary excretion decreased. To address the crucial problem of oligonucleotide degradation, we have investigated the state in which (degraded or not) oligonucleotide was in the plasma and delivered to the liver [10]. It should be pointed out that the evaluation of the in
vivo stability of oligonucleotides represents a major technical difficulty. Two techniques are generally utilized to quantify this degradation: autoradiography and HPLC. None of these methodologies are able to combine a high sensitivity with an accurate quantitative determination of the undegraded oligonucleotide. We have, thus, proposed an original method that consists in the use of a TLC analyser allowing, in polyacrylamide gels, to quantify the amount of undegraded pdT 16 in tissues such as liver and plasma [11]. This method is able to distinguish between oligonucleotides differing by only one nucleotide in length (Fig. 5). Using that methodology, it was found that a significant amount of 33 P-pdT 16 was kept intact in the liver (Fig. 6) and the plasma when administered under the form of nanoparticles [10]. Finally, this system has been proved to be efficient in vivo when applied to an
Fig. 4. Liver (A) and Kidney distribution of 33P-pdT 16 free (s) or associated to PIBCA nanoparticles (d) after IV administration in mice.
Fig. 5. Electrophoretic profile of 15-mer (a), 16-mer (b) and a mixture of both oligonucleotides (c) on a 24 cm high dried gel; from [11].
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E. Fattal et al. / Journal of Controlled Release 53 (1998) 137 – 143
diseases such as viral hepatitis or liver cancers or metastasis.
Acknowledgements This paper has been partly supported by a grant of the CNRS (GDR n8 1207) and by the ARC (Association pour la Recherche contre le Cancer).
References
Fig. 6. Electrophoresis chromatogram of intact pdT 16 in liver 5 min after intravenous administration (5 nmol / kg) of 33P-pdT 16 associated to PIBCA nanoparticles (a) or of 33 pdT 16 free (b). (c) is the control (liver homogenate spiked with 18 000 dpm of intact 33 pdT 16); from [10].
anti-ras oligonucleotide: this system markedly inhibited Ha-ras-dependent tumour growth in nude mice in a highly specific manner [12].
4. Conclusion In conclusion, the results presented in this review show that the association of oligonucleotides with biodegradable PACA nanoparticles is possible if using a hydrophobic counter-ion. This system has been proved to be efficient not only for protecting the oligonucleotide from the degradation by 39-exonucleases but also for increasing the intracellular capture of these molecules. Organ distribution profile of oligonucleotides could be dramatically modified after association with PACA nanoparticles with an increase of liver concentration. These results suggest that nanoparticles might be considered as an interesting carrier system for the antisense treatment of liver
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