Chapter 18 N a n o p a r t i c l e s as a P o t e n t i a l A n t i g e n System
Delivery
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Frank Stieneker and Jörg Kreuter Institut für Pharmazeutische Technologie, Johann Wolfgang GoetheUniversität, Marie-Curie-Strasse 9, D-60053 Frankfurt am Main, Germany
PMMA nanoparticles showed a significant improvement in the induction of the immune response against different antigens such as influenza and inactivated split whole HIV-1 or HIV-2 when compared to other adjuvants. PMMA induced higher titres, which were stable at consistently high levels over prolonged periods of time. The immunization with PMMA was substantially more reproducible and non-responders were not observed. PMMA was biocompatible without any observable side effects or toxic reactions. The titres showed a clear dependence on the hydrophobicity and on the particle size of the respective adjuvant. Comparison of different adjuvants with HIV-2 as the antigen demonstrated that the specificity of the induced antibodies was dependent on the interaction between the various viral proteins and the adjuvant. A large number of antigens such as smaller peptides, virus subunits and genetically engineered proteins are weak immunogens and induce little or no protection. For this reason, many vaccines contain not only the antigen but also adjuvants for enhancing the immune reaction. Until now, a large number of adjuvants have been developed by using mineral compounds (aluminum compounds, bentonite), emulsions (Freund's complete and incomplete adjuvant), surfactants (non-ionic block polymers), peptides (MDP, TMDP), lipids (lipid A), liposomes, colloidal carriers (ISCOMs, nanoparticles), and other compounds. Unfortunately, most of these adjuvants cause toxic side effects or cannot be prepared in reproducible manner. It is known that the particle size of dispersed adjuvants strongly influences the adjuvant effect (i, 2). In particular, slightly different production conditions and aging alter the structure and properties of aluminum compounds (3-6). These production and storage problems may lead to different qualities of alum that show no correlation between the absorption properties of the material and the obtained adjuvant effect (7). To avoid these problems, many different adjuvants were developed with some success. Nanoparticles were developed as an adjuvant and are defined as solid colloidal particles ranging in size from 10 to 1000 nm. They consist of polymeric materials (polyacrylates, polylactides, denatured proteins) in which the active agent (drug or biologically active material) is dissolved, entrapped, encapsulated and/or to 0097-6156/94/0567-0306$08.00/0 © 1994 American Chemical Society In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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which the active agent is adsorbed or attached (8). One attempt was the employment of nanoparticles by Kreuter and Speiser in 1976 (9). One of the most promising polymers for nanoparticulate adjuvants is polymethylmethacrylate ( P M M A ) . This polymer was shown to be slowly biodegradable in the form of nanoparticles without any observable toxic side-effects (10). Because of its high safety, it has been used in surgery for 50 years. In addition, P M M A nanoparticles can be polymerized in a physicochemically reproducible manner within narrow limits (11, 12). Thirdly, it is rather hydrophobic, which, as it will be shown below, enhances the adjuvant effect for a number of antigens.
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Preparation of Nanoparticles Nanoparticles may be prepared from different materials. For the reasons mentioned already, polymethylmethacrylate (PMMA) holds great promise for use as an antigen delivery system. The preparation of P M M A nanoparticles can be carried out either by heat polymerization or by gamma-irradiation (12,13). After purifying the monomer (MMA) from polymerization inhibitors (8, 12), it is dissolved in distilled water or in a desired buffer solution in amounts of up to 1.5%. Polymerization is initiated by irradiation (500 kRad) or by addition of ammonium peroxodisulfate or potassium peroxodisulfate and heating to 65-85 °C. The resulting dispersion can be stored as an aqueous solution or lyophilized powder. Antigen may be incorporated into the particles by irradiation-induced polymerization of the monomer in the presence of the antigen. Although this may lead to the denaturation of many proteins, the antigenicity of the influenza antigens was fully retained (9,14). Alternatively, antigens may be adsorbed onto the surface of the particles by adding antigen to the solution after the polymerization and gently agitating the mixture at 4 °C for 24 hours (14). Depending on the antigen this may lead to the adsorption of practically all antigen (74). Body Distribution and Elimination of PMMA Adjuvant Nanoparticles 14
As previously shown, almost all of the radioactivity of C - l a b e l l e d P M M A nanoparticles stays at the injection site after subcutaneous injection (10, 15). During the first week after the material was injected, only 6% of the radioactivity was eliminated urinarily and only 5% was eliminated fecally. Between the first week and 70 days post administration, the elimination rate decreased to 0.005% per day. The elimination rate stayed at this low level up to two hundred days post administration. Then the elimination of the radioactive material increased exponentially to 1% of the radioactive dose per day up until day 287. Sixty to seventy per cent of the initial radio labelled nanoparticle dose remained at the injection site until day 287 and was not distributed into the residual body. The shape of the elimination rate curve points to the following scenario which was observed previously with other polymers including polylactic acid, and which may be best explained by biodégradation of the polymer. The initial high excretion rate may be due to the elimination of lower molecular weight components of the nanoparticle polymer. After this, between 70 and 200 days, very little nanoparticle material is excreted. Nevertheless, the biodégradation of the P M M A probably occurs during this time similar to other biodegradable polymers. The transport and elimination of this degraded material, however, seems to take place only when a considerably lower molecular weight is reached, thus explaining the steady exponential elimination rate increase after 200 days up to the sacrifice of the animals after 287 days. Parallel to the elimination rate increasem a comparable 100 to 300-fold increase in radioactivity in the body also was observed. Important requirements for the use of a protein or antigen delivery system as an adjuvant for vaccines are the biocompatibilty and the biodegradability of the vaccine
In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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formulation. Histological examination of the injection site of influenza vaccines containing P M M A nanoparticles showed no abnormal tissue response. The physiological reactions of the tissue were the same as those observed after the injection of the control fluid influenza preparation (16). P M M A , therefore, may be better tolerated than other adjuvants, such as aluminum hydroxide, since aluminum compounds are known to induce granuloma (17,18).
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Physicochemical Characterization To characterize the physicochemical properties of the nanoparticles different methods were employed. TTiese techniques include scanning and transmission electron microscopy, photon-correlation spectroscopy, BET-surface area analysis, helium pycnometry, X-ray diffraction, measurement of surface charge, determination of wettability (77) and gel permeation chromatography (79). These techniques and their use are listed in Table I. Influence of Physico-Chemical Properties on the Adjuvant Effect Particle Size. The influence of the particle size of P M M A and of polystyrene nanoparticles on the immune response to influenza and B S A (bovine serum albumin) was determined after intramuscular injection. The particle sizes ranged from 62 nm to 10 μπι. Smaller particles below 350 nm induced a significantly higher immune response (2). The optimal particle size was approximately 100 to 150 nm (2). However, since smaller particles were not thoroughly investigated, it cannot be ruled out that the optimal particle size may be smaller. Hydrophobicity. The influence of the nanoparticle hydrophobicity on the adjuvant effect was investigated by employing B S A and influenza subunits as antigens (20). The hydrophobicity of the particles was altered by varying the functional groups and the side chains of the monomers. These results indicated that the adjuvant effect of the particles increased with increasing hydrophobicity of the polymers (Figure 1). Adjuvant Effects of Nanoparticles with Different Antigens Influenza. In 1976, Kreuter and Speiser (8) described the production of P M M A particles ranging in size from 50 to 300 nm by using γ-irradiation to induce the polymerization of the monomer methylmethacrylate. Adsorption of influenza to the particles led to immune responses which were comparable to those obtained with aluminum hydroxide gel. Polymerization of the particles in the presence of the antigen provided a higher immune response. Similar experiments were performed by using solubilized split influenza antigens, which are weak immunogens. Incorporation, as well as adsorption, of this antigen led to better immune responses than those obtained with aluminum hydroxide adjuvanted vaccines. P M M A induced remarkably high titres with low antigen concentrations (76). These studies also showed an optimal immune response after injection with an adjuvant concentration of 0.5% P M M A nanoparticles. The highest titres were measured 4 weeks after immunization. The greatest differences between aluminum hydroxide and P M M A were observed at low antigen concentrations. In challenge experiments, mice were protected against influenza. Immunization with P M M A provided better protection of mice against the influenza virus than alum immunization. This effect was more pronounced after long time periods (27). In order to determine the storage and heat stability of influenza vaccines, different preparations (PMMA, alum, fluid) were stored at 40 °C for various time periods up to 10 days and were then administered i.m. to mice. The storage at 40 °C
In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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Table I. Physicochemical characterization methods for nanoparticles Parameter
Method
Particle size
Photon correlation spectrometry (PCS) Transmission electron microscopy (TEM) Scanning electron microscopy (SEM) S E M combined with energy-dispersive X-ray spectrometry Scanned-probe microscopes Fraunhofer diffraction
Molecular weight
Gel chromatography
Density
Helium compression pycnometry
Crystallinity
X-ray diffraction Differential scanning calorimetry (DSC)
Surface charge
Electrophoresis Laser Doppler anemometry Amplitude wheighted phase structuration
Hydrophobicity
Hydrophobic interaction chromatography (HIC) Contact angle measurement
Surface properties
Static secondary ion mass spectrometry (SSIMS)
Surface element analysis
X-ray photoelectron spectroscopy for chemical analysis (ESCA)
In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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Prep.Al(OH) (0.2%)
θ
3
P a r t i c l e s i z e (nm) Polymer
103
193 271
Polystyrene
62
132 PMMA
257
9
Fluid
359 201 1941 1:2 VA 2:1 HEMA MMA mol. r a t i o :
Figure 1. Precipitation ring diameters (antibody response; mean ± 95% confidence intervals) after immunization of mice with different bovine serum albumine vaccines, particle sizes of the adjuvant particles, and copolymer composition. P M M A , polymethylmethacrylate; H E M A : M M A , 2-hydroxyethylmethacrylate/methylmethacrylate copolymer.
In Formulation and Delivery of Proteins and Peptides; Cleland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
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resulted in a significant decrease in protection against influenza virus for alum, but there was not a significant decline for P M M A . The protection of mice treated with the stored fluid vaccine was negligible after this preparation was stored for more than 60 hours at 40 °C.
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HIV. Three sets of experiments were performed in order to examine the immune response of mice against HIV-1 and HTV-2. In these experiments, the amount of antigen was optimized, the differences in the rate and extent of the immune reactions against both viruses were determined, and some common adjuvants were compared. Evaluation of the Optimal Antigen Amount. Whole HTV-2 as split antigen was used in three different preparations: antigen dissolved in PBS (fluid), antigen adsorbed to aluminum hydroxide gel (alum), antigen adsorbed onto P M M A nanoparticles (PMMA). Various antigen concentrations ranging from 5 to 50 μg per dose (0.5 ml) were used. The antigen content of each preparation was determined by the Bio-Rad Protein Assay. B S A served as reference solution (22). Antibody titres to the antigen were compared over a time frame of 20 weeks by using an ELISA method (Figure 2a-d) (22, 23). The titres were determined as follows: Four microtiter plates were treated as one unit. The first six rows per plate contained serial dilutions of the test mouse sera, the last two rows of each plate contained the same dilutions of the negative control. The highest dilution of the negative control was defined as the background. The cut off was set at zero after subtracting of the mean absorbance of the background and the 3-fold standard deviation from all measured OD495 values (optical density at 495 nm) (22). During the entire observation period, P M M A nanoparticles provided the best adjuvant effect. Between 4 and 20 weeks, the fluid preparation induced significantly lower titres than the other preparations (Figure 2a-d). The alum preparation yielded titres of intermediate levels. They were significantly lower (p
