Co-polymerised peptide particles (CPP) I: synthesis, characterisation and in vitro studies on a novel oral nanoparticulate delivery system

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Journal of Controlled Release 41 (1996) 271-281

controlled release

Co-polymerised peptide particles (CPP) I: synthesis, characterisation and in vitro studies on a novel oral nanoparticulate delivery system Anya M. Hillery l'a, Istvan Toth b, Andrew J. Shaw c, Alexander T. Florence"'* aCentre for Drug Delivery Research, School of Pharmacy, University of London, 29-39 Brunswick Square, London WCIN lAX, UK bDepartment of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29-39 Brunswick Square, London WCIN lAX, UK CDepartrnent of Toxicology, School of Pharmacy, University of London, 29-39 Brunswick Square, London WCIN lAX, UK

Received 14 June 1995; accepted 23 January 1996

Abstract A novel co-polymeric nanoparticulate drug delivery system (Co-polymerised Peptide Particles: CPP) has been developed as a carrier for the oral uptake of therapeutic peptides. The system was based on the co-polymerisation of the active peptide derivative with n-butylcyanoacrylate (n-BCA), the resulting co-polymer being formulated as nanoparticles. The peptide luteinizing hormone releasing hormone (LHRH) was used as a model drug to investigate the viability of the approach. LHRH was covalently bound to vinylacetic acid and the resulting LHRH-vinylacetate conjugate (3e) and a radiolabel (3c*) were subsequently co-polymerised with n-BCA. The polymerisation reaction conditions were manipulated to exploit the particle-forming properties of n-BCA, so that the co-polymer was prepared as particles of average diameter 100 nm, containing LHRH molecules covalently bound as constituents of the oligomeric chains which formed the particles, rather than physically entrapped or adsorbed. The vinylacetic acid functions as a 'linking acid', allowing the formation of a polymerizable derivative of the peptide; also, the stability of the covalent bond between LHRH and vinylacetic acid ensures that the LHRH remains entrapped, and therefore protected, within the carrier in vivo. The co-polymeric particles were found to be stable in vitro when incubated over a 3 h period in gut luminal contents and mucosal scrapings. Their stability was also demonstrated in fetal calf serum and rat serum. In vitro transport studies using the Caco-2 cell line suggested that absorption in vivo was possible. Keywords: Nanoparticles; Oral drug delivery; Co-polymeric particles; Luteinizing hormone; Releasing hormone

I. Introduction The delivery of therapeutic peptides and proteins via the oral route represents a major challenge. One Corresponding author, Tel: 0171 753 5819; fax: 0171 837 2695. ~Current address: Department of Pharmacy, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK.

approach to enhance the oral uptake of these moieties is to exploit the phenomenon of particulate uptake across the gastrointestinal (GI) tract, using colloidal particles as drug carriers. Such carriers can theoretically protect a labile drug from degradation in the gut, and the systemic delivery of the drug could, in principal, arise from the oral absorption of the intact particle [1]. There is considerable recent evidence supporting the phenomenon of particulate

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A.M, Hillery et al. / Journal of Controlled Release 41 (1996) 271-281

uptake [2-4], and the Peyer's patches of the Gut Associated Lymphoid Tissue (GALT) appear to play an important role in the uptake process [5-8]. The involvement of the GALT in particle uptake has the associated advantage that some particles may subsequently reach the systemic circulation by traversing through lymphatics, thereby avoiding the firstpass effect of the liver. Biodegradable particles that have received attention as carriers for oral drug delivery include polyalkylcyanoacrylate (PACA) nanoparticles [9,10] and nanocapsules [11-14]. PACA nanoparticles are synthesized by the controlled polymerisation of alkyl-2cyanoacrylate monomers, to yield colloidal particles which have the advantages of biocompatibility, biodegradability and mild conditions of synthesis [15]. Other oral biodegradable carriers include poly(lactide) and poly(lactide-co-glycolide) particles [1618], and amino acid 'proteinoids' [ 19]. In all of the particulate delivery systems mentioned, the active drug moiety is physically entrapped or adsorbed to the carrier. Drug loading of PACA nanoparticles is conventionally via adsorption, which is a variable process [15,20,21] that also creates difficulties in separating free from adsorbed drug [22], so that in some cases a suspension containing both free and bound drug has been used [9,23]. A drug moiety can readily desorb from the PACA carrier due to, for example, changes in pH [15], thus a drug which was adsorbed in the acidic conditions of the polymerisation reaction may subsequently desorb in the alkaline environment of the GI tract, so that it is no longer protected by the carrier. The amount of drug which is physically adsorbed may be reduced because the drug moiety per se, rather than hydroxyl ion, may initiate alkyl-2cyanoacrylate polymerisation. This results in a percentage of the drug becoming inadvertently covalently bound to isohexyl- and isobutyl-cyanoacrylate particles [20,24]. PACA nanoparticles contain surfactant as a stabilizer, the removal of which results in irreversible particle aggregation [25,26]. Thus conventional methods of latex purification (e.g. dialysis, ion exchange, ultra-centrifugation) are not possible, and purification is limited to simple filtration through a sintered glass funnel [15]. This process does not eliminate contaminants which include unreacted

monomer and soluble oligomeric ions [27]. Another approach to promote oral absorption, enhance circulating half-life, or achieve drug targeting, is the formation of a drug-polymer conjugate, e.g. conjugation of the active drug moiety with polyethylene glycol [28-30] or hydroxypropylmethacrylamide [31,32]. We describe here a novel approach that combines both methods of oral absorption enhancement, i.e. the use of particulate carriers and the use of drugpolymer conjugates, by synthesizing a drug-polymer conjugate that formed its own delivery system, being formulated as nanoparticles in which the drug moiety was covalently bound, rather physically entrapped, within the system. By forming a co-polymer delivery system using n-butylcyanoacrylate (n-BCA) as one of the monomers, the particle-forming properties of the alkyl-2-cyanoacrylates could be exploited, so that the drug-polymer conjugate was formulated as nanoparticles. Luteinizing hormone releasing hormone (LHRH), a decapeptide synthesized in the hypothalamus which controls the release of luteinizing hormone and follicle stimulating hormone, was chosen as a model peptide to investigate the feasibility of the approach. Either an irreversible or reversible (i.e. a pro-drug linkage) covalent bond could have been used as the drug-matrix linkage. To ensure that LHRH remained bound, and therefore protected, by the carrier in vivo, a relatively stable covalent bond was deemed to be required. Studies on amide bonds as drug-matrix linkages have determined that these bonds were little cleaved in the absence of a 'spacer' bridging group and when a spacer was present, the bonds were cleaved to a small extent inside lysosomes, but were stable in the GI tract and the bloodstream [33], therefore the amide bond was selected as the drug-matrix linkage. We describe here the synthesis and characterization of the novel co-polymeric particles, investigations on their in vitro stability in gut contents, mucosal scrapings and serum, and in vitro transport studies using the Caco-2 cell culture model. The Caco-2 cell line differentiates to cells with an enterocyte-like morphology under standard cell culture conditions, forming confluent monolayers [34] and has been used successfully to model the intestinal absorption of drugs [35-37].

A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281


2. Materials and methods

2.2. Peptide purification

2.1. Solid phase peptide synthesis

Preparative and analytical HPLC was carried out using an Applied Biosystems 400 Solvent Delivery System and Injector Mixer, with solvent gradients effected by two micro-processor-controlled Gilson 302 single piston pumps. HPLC grade solvents, 0.1% T F A / H 2 0 (A) and 80% acetonitrile/0.1% T F A / H 2 0 (B), were filtered under vacuum through a 25 /zm filter prior to use. Compounds were detected with an Applied Biosystems Programmable Absorbance Detector at 214 nm (analytical) and 230 nm (preparative), and chromatographs were recorded with an LKB 2210 single channel chart recorder. Preparative HPLC was carried out on a TSK-gel preparative column (2 cm x 30 cm), with a solvent gradient that increased from 0-100% solvent B (80% acetonitrile) over a period of 150 min, with a constant flow rate of 9 ml/min. The purity of the LHRH-vinylacetate conjugate 3e was established by analytical HPLC on a Beckman Ultrasphere Octyl column (5 /zm, 4.6 mm X 25 mm) with guard column (5/zm, 4.6 mm X 45 mm) and a solvent gradient that increased from 0-100% solvent B (80% acetonitrile), over a period of 30 min, with a constant flow of 0.7 ml/min. 3e: MS m/z (%): 1269 [M+H] + (100), 1225 (4), 1071 (5), 716 (10), 661 (12). R t = 14 min.

LHRH was synthesized by a step-wise solid phase procedure [38] on a p-MBHA Novabiochem resin (1 g, substitution 0.48 m m o l / g resin) using an Applied Biosystems 430A Automated Synthesizer and the following N-a-Boc protected, side chain protected amino acids: Boc-Gly, Boc-Pro, Boc-Arg(Tos)-OH, Boc-l_eu-OH-H20, Boc-Gly, Boc-Tyr(2-Br-Z)-OH, Boc-Ser(Bzl)-OH, Boc-Trp(For)-OH, BocHis(DNP)-OH-IPE, Boc-Glu-(OBzl)-OH. Each coupling was achieved using a 4 M excess of preformed symmetrical anhydride of the N-a-Boc protected amino acids in dichloromethane (DCM), or with 1-hydroxybenzotriazole (HOBt) in DCM. A second coupling was carried out in DCM, dimethylformamide (DMF) or HOBt in DMF. After the second coupling, deprotection of the N-termini was performed in 65% trifluoroacetic acid (TFA) in DCM (20 ml for 1 min, then another 20 ml for 10 min). The deprotected peptide-resin was neutralized with 10% diisopropylethylamine (DIEA) in DCM (20 ml for 1 min, twice). The peptide-resin was washed between and after the deprotection and neutralization steps. After chain-assembly of LHRH was completed and the N-terminus deprotected and neutralized, vinylacetic acid (2 mmol, 0.0172 ml) was manually conjugated with the resin-bound peptide. The conjugation was carried out in DCM (30 ml) with dicyclohexylcarbodiimide (DCC) in DCM (2 mmol, 2 ml) and was followed by a second coupling performed in DMF with the addition of HOBt in DCM (1 mmol, 2 ml). In all couplings, the coupling yields were -->99.98% as indicated by quantitative ninhydrin testing. The DNP protecting group from the His was removed with 20% mercaptoethanol-5% DIEA in DMF, and the CHO group from the Trp with 10% piperidine in DMF. After washing with DCM, the peptide-resin was dried overnight with KOH. The peptide was removed from the resin support using the high HF method (1.5 ml cresol, 1.5 ml thiocresol, 20 ml HF) to yield the crude LHRHvinylacetate conjugate 3e, which was precipitated with ether and redissolved in 40% acetic acid (1.5 ml)/glacial acetic acid (10 ml).

2.3. Conjugation reactions Conjugates l a - l f , 2a, 2b and 3 a - 3 d were synthesized in solution according to classical peptide synthetic methods, using carbodiimides, 'active esters' or symmetrical anhydrides [39]. Experimental and physico-chemical data are given for selected compounds pertinent to the synthesis of CPP particles, the remaining data being available on request.

2.3.1. Methyl 2-vinylacetylamido-3phenylpropionate (3c) Vinylacetic acid (0.4 g, 4.6 mmol), phenylalanine methyl ester hydrochloride (1 g, 4.6 mmol), triethylamine (0.42 g, 4.1 mmol), and 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDAC) (0.88 g, 4.6 mmol) were stirred in DCM (30 ml) for 1 h at 0°C, then further EDAC (1.02 g, 5.53 mmol) was added and the reaction stirred for 2 h at room


A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281

temperature. The reaction mixture was washed with 5% HC1 (2 × 20 ml), water (20 ml), dried (MgSO4) and the solvent removed in vacuo. The crude reaction product was purified by TLC. Rf = 0.82 (CH2C12:MeOH 10:1 v/v). Yield 0.9886 g (70.6%) 3e - - 1H NMR (CDC13): o- = 7.40, 7.25 (4H, ab, 4 aromatic H), 7.40 (1H, d, aromatic H), 6.45 (1H, m, CH2=), 6.05, 5.35 (2H, 2m, CH2=), 5.30 (1H, m, NH), 5.02 (1H, m, a-CH), 3.85 (3H, s, COOCH3), 3.30, 3.23 (2H, 2m, CH2-Ar), 3.14 (2H, d, CH2CO ) - - MS m/z: (%) = 270 [M+Na] + (100), 120 (10), 248 (7). 2.3.2. Methyl [2,6-3H]phenylpropionate (3c*) This compound was prepared as described for 3c, after initially preparing the methyl ester of [2,63H]phenylalanine by heating it with thionyl chloride and in methanol. Rf [2,6- 3H]phenylalanine hydrochloride = 0.21, Rf [2,6-3H]phenylalanine methyl ester = 0.58, Rf 3e* = 0.82 (CHzClz:MeOH 10:1 v/v).

2.4. Polymerisation reactions 2.4.1. n-BCA polymer n-BCA monomer (0.5 ml) was added drop-wise under vigorous mechanical stirring to 50 ml of a polymerisation medium consisting of water (45 ml), poloxamer 188 (0.2%), adjusted to pH 3.2 with dilute HC1. The reaction mixture was stirred for 2 h to accomplish polymerisation, then filtered through a sintered glass filter (pore size: 9-15 /xm). Laser Desorption MS: most intense peak at 447. PCS: z average 80 nm, polydispersity 0.15. 2.4.2. Co-polymerisations In the general method for co-polymerisation of n-BCA with acrylic, vinylbenzoic or vinylacetic acid, the n-BCA monomer (0.6 ml) was mixed with the respective acid (0.6 ml or 0.6 g) and the resulting solution (1 ml) was added dropwise to the polymerisation medium described above. For the copolymerisation of n-BCA with compounds ( l a - l f , 2a, 2b, 3a-3d), the respective conjugate (5 rag) was mixed with n-BCA monomer (0.5 ml) and the mixture added dropwise to the polymerisation medium described above.

2.4.3. CPP system (i) LHRH-vinylacetate 3e (2 mg, 0.0015 mmol) was dissolved in H20 (1 ml) and conjugate 3e* (2 mg, 0.007 mmol) was dissolved in n-BCA monomer (0.5 ml). The solutions were added separately and simultaneously to the polymerisation medium (50 ml) described above, adjusted to pH 3.5. The copolymer precipitated immediately on the stainless steel stir-bar. After stirring for 1 h, the polymerisation medium was removed and discarded. The stir-bar, with the adhering precipitated polymer, was dried under vacuum for 24 h and then placed in DMF (2 ml) and stirred until the precipitate had dissolved (8 h). Polymerisation medium (48 ml) was added to the dissolved polymer under vigorous stirring, to form polymeric particles. (ii) The same reaction was carried out using 3e (20 mg, 0.015 mmol) and 3e* (20 mg, 0.07 mmol). ~H NMR (methyl sulphoxide): o - = 8.4-6.6 (m, aromatic H), 4.5, 3.55 (m, CONH), 3.5 (m, butyl-OCH2), 2.5 (m, CH2CO ), 1.6, 1.4, 1.3 (m, polymer chain CHz, CH), 0.9 (m, butyl-CH 2, -CH3). Laser Desorption MS: most intense peak = 2808. PCS: z average 100 nm, polydispersity 0.1. 2.5. Characterization 2.5.1. 1H NMR and MS ~H NMR were obtained with Varian XL-300 and Bruker AM 500 instruments operating at fields of 300 and 500 MHz respectively. Chemical shifts are reported in ppm downfield from internal TMS. Mass spectra were run on a VG analytical ZAB-SE instrument~ using the fast-atom bombardment (FAB) technique with 20-kV Cs + ion bombardment and 2 /xl of an appropriate matrix (3-nitrobenzyl alcohol/ MeOH or thioglycerol with NaI/MeOH. Laser Desorption Mass spectra (LD MS) were run on a Kratos Analytical Kompact Maldi 3 Matrix Assisted Laser Desorption Time of Flight Mass Spectrometer operating in linear mode using a 337 nm laser. 2.5.2. TLC Reaction progress was monitored by thin-layer chromatography (TLC) on Kieselgel PF254 (Sigma, Dorset, UK) using the following solvent systems: (1) DCM, (2) D C M / M e O H 10:0.1 (v/v), (3) DCM/ MeOH 10:1 (v/v). Purification was achieved by TLC

A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281


using Kieselagel PF254+366 o n 20 cm × 20 cm plates, of thickness 1.5 mm, with the aforementioned solvent systems. Reaction progress of tritiated compounds was monitored using a Tracemaster 20 Automatic TLC Linear Analyzer (Berthold Instruments Ltd., St. Albans, UK).

degradation studies, CPP particles (0.2 ml) were added to 20 ml of either fetal calf serum (Gibco, Paisley, UK) or rat serum at 37°C, samples were taken over a 25 h period and analyzed as described for the GI tract study, using TFA-deproteinized serum for controls.

2.5.3. Particle characterization Particle size was determined by photon correlation spectrometry, using a Multi 8 Computing Correlator Type 7032CE with Liconix H e / C d Laser (Malvern, Worcs, UK). Transmission electron microscopy (TEM) was carried out on a Phillips XL 20 Scanner (Phillips, Cambridge, UK), using negative staining with 1% phosphotungstic acid.

2. 7. In vitro transport studies using Caco-2 cells

2.6. In vitro degradation studies The luminal contents and intestinal mucosal scrapings of 4 male Wistar rats (260-270 g, 9 weeks old) were pooled, diluted 1:4 with ice-cold phosphate buffered saline (PBS, pH 7.4) and homogenized using a Potter Elvejem. CPP particles (0.2 ml) were incubated at 37°C with aliquots (0.4 ml) of the intestinal samples and at intervals (10 min, 30 min, 60 min, 90 min and 120 min) aliquots were deproteinized using TFA (10 /zl) and centrifuged at 80 000 × g for 3 h at 4°C. Radioactivity in the supernatant was measured by scintillation counting (Beckmann LS5000CE Liquid Scintillation System). Control samples (t = 0) were prepared by the addition of CPP particles to TFA-deproteinized intestinal samples, followed by centrifugation and scintillation analysis of the supernatant. The percentage degradation was calculated as: (%) Degradation Supernatant disintegrations / min "~ =




The 'Total disintegrations/min' was obtained by scintillation counting of a non-centrifuged control. Results were expressed as the (%) degradation from the T = 0 value (Intact Particles). Further studies were carried out in which the ratio of CPP particles to intestinal samples was 1:5 and 1:20. In the serum

Caco-2 cells were obtained from the European Collection of Animal Cell Culture (Wiltshire, UK). Cells were expanded in flasks with 150 cm 2 growth area at 37°C, 5% CO 2, in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 1% non-essential amino acids and 50 /zg/ml gentamycin. Cells of passage 30 were used. For subculture, confluent cultures were split using 0.25% trypsin in PBS. Cells were added to the inserts of Transwell T M chambers (Transwell, Costar, Bucks, UK), culture plates comprising of 24 wells, containing polycarbonate inserts of 6.5 mm diameter, 0.4/~m pore size and seeded at a density of 2 × 10 6 cells/cm:, as determined by a Neubauer haemocytometer. The integrity of the cell monolayers was tested by the measurement across monolayers and cell-free inserts of (i) the transepithelial electrical resistance (TER) using a WPI EVOM mV/ /2 meter calibrated with 10 and 100 mM KC1 and (ii) transport of [14C]polyethyleneglycol ([IaC]PEG; Amersham, Bucks, UK). The CPP transport experiments were carried out at 37°C on cell monolayers which had been cultivated for 18 days. CPP particles were diluted 5- fold in medium and added in triplicate to the apical side of the cell monolayers. After either 4 h or 6 h incubation periods, samples were withdrawn from both the apical and basal chambers, and the inserts were dissected and washed. The radioactivity in the apical and basal samples and associated with the inserts was determined by scintillation counting. A 6-h transport experiment was also carried out in a modified culture medium, which did not contain FCS. Control experiments were carried out in which the transport of particles was measured across cell-free inserts, after 4 h and 6 h incubation periods. Results were corrected for dilution factors. The percentage of the radiolabel transported to the basal layer was calculated as:

A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281


(%) Transport = 100 Basal chamber disintegrations/min Apical chamber disintegrations/min + Basal chamber disintegrations/min + Filter disintegrations/min


3. Results and discussion

The alkyl-2-cyanoacrylates co-polymerise with a wide variety of compounds, including unsaturated acids [40], therefore our initial strategy was to prepare a polymerizable derivative of LHRH by conjugating the peptide with an unsaturated acid, and then co-polymerizing this conjugate with n-BCA. Acrylic, vinylacetic and vinylbenzoic acids were examined for their suitability in the conjugation and co-polymerisation reactions. The respective acids were co-polymerised with n-BCA and the relative reactivity and rate of polymerisation was determined as: acrylic acid > vinylacetic acid > vinylbenzoic acid. The ~H NMR spectra of the respective copolymers showed the absence of unsaturated protons, indicating that none of the starting monomers were present and that complete co-polymerisation had taken place. The co-polymeric particles were the same size ( - 1 0 0 nm diameter) as particles formed using the n-BCA homo-polymer, as demonstrated by PCS. Various model compounds were conjugated with the unsaturated acids, resulting in acrylic acid conjugates l a - l f , vinylbenzoic acid conjugates 2a and 2b, and vinylacetic acid conjugates 3a-3d. These conjugates were then co-polymerised with n-BCA. The use of acrylic acid was limited by its high reactivity and tendency to auto-polymerise. The conjugation of acrylic acid with the model compounds alanine ethyl ester, phenylalanine methyl ester, and tetradecylamine resulted in the formation of polymeric products including dimers, trimers, tetramers and pentamers. In the co-polymerisation reactions of acrylic acid conjugates l a - l f with nBCA, extensive pre-polymerisation took place in situ on mixing of the respective conjugates l a - l f with n-BCA, prior to the addition of the mixture to the 'particle-forming' polymerisation medium. The use of vinylbenzoic acid was limited by its poor solu-

bility in the solvents used for the conjugation reactions. For the co-polymerisation reactions, vinylbenzoic acid conjugates 2a and 2b were poorly soluble in n-BCA monomer. Vinylacetic acid was sufficiently reactive for satisfactory conjugation with model compounds (dopamine, phenylalanine methyl ester, [3H]phenylalanine methyl ester and 4-aminofluorescein) and the conjugates 3a, 3b and 3c copolymerised satisfactorily with n-BCA, forming copolymeric particles of similar size to n-BCA particles. The ~H NMR spectra of the co-polymers of n-BCA with 3a, 3b and 3c showed the absence of unsaturated hydrogens and the correct proton ratio of the copolymerised compounds. The conjugation and co-polymerisation reactions demonstrated that vinylacetic acid was the most suitable unsaturated acid for the preparation of a polymerizable derivative of LHRH. The peptide was synthesized on solid-phase, using glutamic acid (rather than pyroglutamic acid) at the N-terminus. In a novel procedure, vinylacetic acid was conjugated with the free amino group on the N-terminus, while the chain-assembled peptide was still side-chain protected and bound to the resin support. This procedure had the advantage that none of the reactive side-chain functionalities present on LHRH could interfere with the conjugation reaction, because they were blocked by protecting groups. Furthermore, auto-polymerisation of the LHRH-vinylacetate conjugate 3e was prevented because the conjugate was anchored to the resin support. Vinylacetic acid was also conjugated with tritiated phenylalanine methyl ester, forming conjugate 3c*, to serve as an in vivo marker for the system. Thus the radiolabel and LHRH were conjugated to the particles via vinylacetic acid, using the same type of stable covalent bond, and both moieties were protected from degradative effects within the particulate matrix. This suggested that the radiolabel and LHRH should remain covalently bound within the particles and that analysis of radioactivity after oral dosing would be indicative of the presence of both LHRH and nanoparticles. 3.1. CPP system

The three monomers, n-BCA, LHRH-vinylacetate 3e, and compound 3c*, were co-polymerised. The

A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281 m

f 1


JlN H" rn




Fig. 1. Structure of CPP co-polymericparticles: [n-BCA]-[LHRHvinylacetate]-[N-vinylacetyl-3H-Phe-OCH3]. polymerisation conditions were manipulated so that initially the co-polymer precipitated out of the reaction. After decantation of the aqueous phase, the precipitated co-polymer on the stir-bar was dried, dissolved in DMF and subsequently recovered as

Fig. 2. Transmission electron micrograph (×67 500) of CPP particles.

Fig. 3. Transmission electron micrograph (×97 300) of CPP particles.


nanoparticles, by the addition of 'particle-forming' polymerisation medium (see Section 2). The approach used in this work is novel, in that it involved the co-polymerisation of a peptide derivative with other monomers, formulated so that the co-polymer formed its own particulate delivery system. The active drug molecules were constituents of the oligomeric chains which formed the particles and were covalently bound to the system. Thus the delivery system comprised of co-polymerised peptide particles (CPP) (Fig. 1). The particles had a size of - 1 0 0 nm, as determined by PCS and verified by TEM (Fig. 2 and Fig. 3). Molecular weight determination using gel permeation chromatography (GPC) was used [41] to determine polymer molecular weight of isobutyl- and isohexyl-cyanoacrylate polymers, and the authors found that the nanoparticles were comprised of shortchain oligomers, having a molecular weight between 500 and 1000. We report here similar findings of low molecular weight chains constituting the CPP system, using the simple and rapid technique of laser desorption MS. The laser desorption MS showed that the molecular weight distribution of the particles followed a Gaussian curve with the most intense peak at 2808 (Fig. 4). The mass difference between the peaks of the CPP laser desorption mass spectrum was 152, which corresponded to the molecular weight of the n - B C A monomer. As the number of monomers in the CPP formulation increased, the most intense peak in the laser desorption mass spectrum increased proportionally, from 447 for the n-BCA homo-polymer, to 1346 for the n-BCA-radiolabel co-polymer; and finally 2808 for the CPP system (Table 1). As described above, unreacted monomers and soluble oligomeric ions represent a serious contaminant of PACA particle syntheses that cannot be removed by conventional methods of latex purification. In the synthesis of CPP particles, the co-polymer [n-BCA-3e-3e*] precipitated out of the reaction, leaving the unreacted n - B C A monomers in an aqueous phase, which was discarded. The CPP particles were subsequently recovered from the precipitated co-polymer. The absence of monomers in the CPP system was verified by the absence of unsaturated hydrogens in the 1H NMR spectrum of the particles. When 20 mg 3e, 20 mg 3e* and 500 mg n-BCA


A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281 Run: SKB tint


3 9 SLIp 92 14:23 IGT/AH ; 21 I V [ s u m - 1 0 4 0 mVl

6inaptntc r=hot n o s .

acid 1-49



100 95 90"




2350 2196

7o -: 5o .i



5 0 -"


1890 2( 3

45 40




3 0 -!








.1(132 43.40 4a')2







Fig. 4. Laser desorption mass spectrum of CPP particles.

were used in the co-polymerisation, the ratio of the aromatic protons and the O - C H 2 protons in the co-polymer was 1:5, indicating the presence of 4% L H R H and 4% phenylalanine in the particles, confirming complete co-polymerisation. When the ratio of n-BCA to LHRH-vinylacetate and to the radiolabel was 250:1, N M R structure identification was not feasible, however the radioactivity was measured and 98% of the initial radioactivity was detectable in the CPP particles, confirming that complete co-polymerisation of the respective monomers occurred. Peptide integrity in the CPP system was confirmed using a double-antibody radio-immunoassay (unpubTable 1 Most intense peak in the laser desorption mass spectra Sample n-BCA homopolymer (A) [n-BCA]-[N-vinylacetyl-3H-Phe]co-polymer(A + B) CPP particles (A + B + C)


447 1346 2808

lished results) and the biological activity of the peptide was demonstrated after oral dosing with the CPP system to rats (unpublished results).

3.2. In vitro d e g r a d a t i o n s t u d i e s

No degradation of the CPP particles was detected when they were incubated in a 1:2 or 1:5 ratio with a mixture of gut contents and surface mucosal scrapings. When the ratio of CPP particles to intestinal sample was increased to 1:20, the radioactivity detected in the supernatant increased, indicating some degradation of the particles into water-soluble oligomers. However only - 8 % of the total radioactivity was detected in the supernatant over a 2 h incubation period (Fig. 5). As the 1:20 incubation ratio represented a higher ratio than likely in vivo, these in vitro experiments suggested the particles would remain predominantly intact in the GI tract after oral dosing. The CPP particles also demonstrated stability in both FCS and rat serum, with only


A.M. Hillery et al. I Journal of Controlled Release 41 (1996) 271-281

Table 2 Percentage transport of radiolabel across Caco-2 monolayers

Intact Particles 100 T ~ - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .




60 Time





Fig. 5. Percentage degradation of CPP particles on incubation of the particles in a 1:20 ratio with a gut contents/mucosal scrapings mixture.

- 1 5 % degradation detected after 15 h incubation, rising to 20% over a 25 h period (Fig. 6). 3.3. Caco-2 monolayer transport studies

Transepithelial resistance (TER) measurements and transport of [laC]polyethyleneglycol ([14C]PEG) were significantly different across monolayers than across cell-free inserts (P < 0.1 and P < 0.001 respectively), which confirmed the integrity o f the monolayers and demonstrated that the diffusion barrier was provided by the monolayers, rather than the inserts supporting them. Twenty-five percent and 29% o f the CPP radiolabel was transported from the apical to the basal side of the monolayers after 4 h

Ifltact Particles 100 ~ll~- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .



Incubation time and medium

% Transport

4 6 6 4 6

25.2 _+3.1 28.9 _+3.1 28.9 -+ 0.4 65.3 -+ 1.3 72.3 +_ 1.7

h h h h h

(medium + FCS) (medium + FCS) (medium - FCS) (cell free inserts) (cell free inserts)

and 6 h incubation, respectively (Table 2), which suggested that absorption in vivo might be possible. For all samples, adsorption of radiolabel to the monolayer/inserts was negligible. FCS present in the culture medium represented an additional source of esterase activity not found in the GI tract, therefore the FCS may have been responsible for accelerated particle degradation in this in vitro model. However the percentage transport of radiolabel in an FCSdepleted medium (Medium-FCS, Table 2) was similar to that determined in a medium containing FCS. The results suggested that transport in vivo might take place, although this study measured the transport of the radiolabel across the monolayers, rather than the proof of transport of intact peptide or particles.

4. Conclusions A novel drug delivery system (CPP) was developed for the oral delivery of peptides. An [LHRH derivative/n-BCA/radiolabel] co-polymer was formulated as nanoparticles, containing covalently bound L H R H molecules as constituents of the oligomeric chains which formed the particles. Stable nanoparticles of - 1 0 0 nm diameter were prepared and the particles were stable in vitro when incubated in luminal contents, mucosal scrapings and serum. It was concluded that the particles demonstrated potential as an oral drug delivery system.


Acknowledgments 0






Time (h}

Fig. 6. Percentage degradation of CPP particles, when incubated with fetal calf serum (A) or rat serum (m).

We thank Professor Calum Macfarlane for his interest and for the support of A.M.H. We express our gratitude to Mr. Bob Lambert and Dr. Alan


A.M. Hillery et al. / Journal of Controlled Release 41 (1996) 271-281

Lennox, Loctite Ireland Ltd., for advice and generous supply of the monomers.

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