Gelatin microspheres and sponges for delivery of macromolecules

Gelatin microspheres and gelatin sponges were prepared by coacervation and freeze drying techniques, respectively. Both systems were crosslinked with glutaraldehyde. The mean diameter of the microspheres were in the range of 40-80 microm and the mean pore size of the sponges was 130-220 microm depending on the preparation conditions. Bovine serum albumin (BSA) was added into the preparation solutions and entrapped in the microspheres and sponges. BSA addition to sponges was also achieved by addition of BSA-containing microspheres into the sponges. The release kinetics of BSA from the prepared systems were examined. Studies demonstrated that release is dependent on the amount of BSA present in the system and crosslinking densities of microspheres. It was concluded that gelatin microspheres and gelatin sponges are promising carrier matrices for macromolecules.     

Controlled release systems for proteins based on gelatin microspheres

The preparation and characterization of biodegradable gelatin microspheres for the controlled release of peptides and proteins has been investigated. Bovine serum albumin (BSA) was chosen for incorporation into the gelatin microspheres and the spheres were characterized for the in vitro release of BSA and other properties. BSA was labelled with fluorescein isothiocyanate (FITC) for easy analysis. FITC-BSA was entrapped into the gelatin microspheres using a polymer dispersion technique developed in our earlier studies. The morphological characteristics of microspheres were analysed by optical and scanning electron microscopy (SEM). The optical and SEM photographs of FITC-BSA microspheres showed the solid spherical nature of the spheres. The entrapment efficiency of FITC-BSA was about 62%. The in vitro release pattern of FITC-BSA showed that 51 % of the entrapped drug was released during the first day and the release followed approximate zero order kinetics from day 2 onwards. The total release of FITC-BSA lasted for about 8 days. SDS-PAGE analysis revealed that BSA was not degraded by this preparation of microspheres.     

Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs

Bovine serum albumin (BSA) and diphtheria toxoid (DT) were loaded by passive absorption from aqueous solutions into preformed glutaraldehyde cross-linked chitosan microspheres. In vitro release of BSA under sink conditions at 37°C showed that even though there was a large burst effect, there was a more or less steady increase with time thereafter for several days. Coating the BSA-loaded particles with paraffin oil or with a polymer, such as polylactic acid, modulated drug release. After the initial burst from PLA coated particles, the release rate increased with time for nearly 2 months. Preliminary immunogenicity studies on Wistar rats using DT loaded chitosan spheres showed that the antibody titres were fairly constant over a 5-month period, although very low compared to DT given on alum as control. Histological studies of placebo microspheres intramuscularly injected into rats demonstrated their tissue compatibility. Biodegradation was not complete in 6 months demonstrating the potential of cross-linked chitosan spheres as a long-acting drug delivery vehicle. The study demonstrated the possibility of incorporating biological macromolecules which are very sensitive to organic solvents, pH, temperature, ultrasound, etc. by a passive absorption technique to degradable biopolymer matrices thereby preserving their biological integrity. It is also shown that drugs passively absorbed into such matrices by taking advantage of their swelling behaviour need not necessarily be released completely in the initial 'burst' and a sustained release may be possible for macromolecules thus incorporated.     

Novel polyelectrolyte complexes based on poly(methacrylic acid)-bis(2-aminopropyl)poly(ethylene glycol) for oral protein delivery

In the present investigation a simple and effective strategy was employed for the development of pH-sensitive self-assembling microparticles based on poly(methacrylic acid) (PMAA)-bis(2-aminopropyl)poly(ethylene glycol) (APEG), and their efficiency in oral protein delivery was evaluated. An inter-ionic gelation process was employed for the preparation of microparticles and particles were obtained spontaneously during the process without using any surfactants or stabilizers. Particle size analysis was carried out to measure average particle size and surface morphology was evaluated using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was incorporated onto these microparticles to evaluate the loading and release properties of the matrix. PMAA-APEG microparticles displayed pH responsive release profile, as less than 10% of encapsulated BSA was released at pH 1.2 in 2 h and more than 80% of loaded protein was released within 3 h at pH 7.4. Carboxymethyl beta-cyclodextrin (CM beta CD)-insulin non-covalent inclusion complex was prepared to enhance the stability of insulin formulations and complex formation was analyzed by fluorescence spectroscopic studies. CM beta CD-complexed insulin was encapsulated into PMAA-APEG microparticles by a diffusion filling method and biological activity of entrapped insulin was evaluated using an ELISA. Finally mucoadhesive studies of PMAA-APEG microparticles were carried out on freshly excised rat intestinal mucosa at neutral pH to establish the adhesive nature of the material.     

Novel polyelectrolyte complexes based on poly(methacrylic acid)-bis(2-aminopropyl)poly(ethylene glycol) for oral protein delivery

In the present investigation a simple and effective strategy was employed for the development of pH-sensitive self-assembling microparticles based on poly(methacrylic acid) (PMAA)-bis(2-aminopropyl)poly(ethylene glycol) (APEG), and their efficiency in oral protein delivery was evaluated. An inter-ionic gelation process was employed for the preparation of microparticles and particles were obtained spontaneously during the process without using any surfactants or stabilizers. Particle size analysis was carried out to measure average particle size and surface morphology was evaluated using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was incorporated onto these microparticles to evaluate the loading and release properties of the matrix. PMAA-APEG microparticles displayed pH responsive release profile, as less than 10% of encapsulated BSA was released at pH 1.2 in 2 h and more than 80% of loaded protein was released within 3 h at pH 7.4. Carboxymethyl β-cyclodextrin (CMβCD)-insulin non-covalent inclusion complex was prepared to enhance the stability of insulin formulations and complex formation was analyzed by fluorescence spectroscopic studies. CMβCD-complexed insulin was encapsulated into PMAA-APEG microparticles by a diffusion filling method and biological activity of entrapped insulin was evaluated using an ELISA. Finally mucoadhesive studies of PMAA-APEG microparticles were carried out on freshly excised rat intestinal mucosa at neutral pH to establish the adhesive nature of the material.     

Modulation of protein delivery from modular polymer scaffolds

Growth factors are increasingly employed to promote tissue regeneration with various biomaterial scaffolds. In vitro release kinetics of protein growth factors from tissue engineering scaffolds are often investigated in aqueous environment, which is significantly different from in vivo environment. This study investigates the release of model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA) from various scaffolding surfaces and from encapsulated microspheres in the presence of ions, proteins, and cells. The release kinetics of proteins in media with varying concentrations of ions (NaCl) suggests stronger electrostatic interaction between the positively charged histone with the negatively charged substrates. While both proteins released slowly from hydrophobic PCL surfaces, plasma etching resulted in rapid release of BSA, but not histone. Interestingly, although negatively charged BSA released readily from negatively charged collagen (col), BSA released slowly from col-coated PCL scaffolds. Such electrostatic interaction effects were abolished in the presence of serum proteins and cells as evidenced by the rapid release of proteins from col-coated scaffolds. To achieve sustained release in the complex environment of serum proteins and cells, the model proteins were encapsulated into poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within col-coated PCL scaffolds. Protein release from microspheres was modulated by changing the lactide-to-glycolide ratio of PLGA polymer. BSA adsorbed to col released faster than histone encapsulated in microspheres in the presence of serum and cells. Collectively, the data suggest that growth factor release is highly influenced by scaffold surface and the presence of ions, proteins, and cells in the media. Strategies to deliver multiple growth factors and studies which investigate their release should consider these important variables.     

PLGA microsphere/calcium phosphate cement composites for tissue engineering: in vitro release and degradation characteristics

Bone cements with biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have already been proven to provide a macroporous calcium phosphate cement (CPC) during in situ microsphere degradation. Furthermore, in vitro/in vivo release studies with these PLGA microsphere/CPC composites (PLGA/CPCs) showed a sustained release of osteo-inductive growth factor when drug was distributed inside/onto the microspheres. The goal of this study was to elucidate the mechanism behind drug release from PLGA/CPC. For this, in vitro release and degradation characteristics of a low-molecular-weight PLGA/CPC (M(w) = 5 kg/mol) were determined using bovine serum albumin (BSA) as a model protein. Two loading mechanisms were applied; BSA was either adsorbed onto the microspheres or incorporated inside the microspheres during double-emulsion. BSA release from PLGA microspheres and CPC was also measured and used as reference. Results show fast degrading polymer microspheres which produced a macroporous scaffold within 4 weeks, but also showed a concomitant release of acidic degradation products. BSA release from the PLGA/CPC was similar to the CPC samples and showed a pattern consisting of a small initial release, followed by a period of almost no sustained release. Separate PLGA microspheres exhibited a high burst release and release efficiency that was higher with the adsorbed samples. Combining degradation and release data we can conclude that for the PLGA/CPC samples BSA re-adsorbed to the cement surface after being released from the microspheres, which was mediated by the pH decrease during microsphere degradation.     

pH-sensitive behavior of two-component hydrogels composed of N,O-carboxymethyl chitosan and alginate

A two-component pH-sensitive hydrogel system composed of a water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC) and alginate cross-linked by genipin, glutaraldehyde or Ca2+ was investigated. Preparation and structures of these hydrogels and their swelling characteristics and release profiles of a model protein drug (bovine serum albumin, BSA) in simulated gastrointestinal media are reported. At pH 1.2, the swelling ratios of the hydrogels cross-linked by distinct methods were limited. Of note is that the lowest swelling ratios of test hydrogels were found at pH 4.0. At pH 7.4, the carboxylic acid groups on test hydrogels became progressively ionized and led to a significant swelling. There was barely any BSA released from the glutaraldehyde-cross-linked hydrogel throughout the entire course of the study. The amounts of BSA released at pH 1.2 from the genipin- and Ca(2+)-cross-linked hydrogels were relatively low (approx. 20%). At pH 4.0, there was still significant BSA release from the Ca(2+)-cross-linked hydrogel, while the cumulative BSA released from the genipin-cross-linked hydrogel was limited due to its shrinking behavior. At pH 7.4, the amount of BSA released from the genipin- and Ca(2+)-cross-linked hydrogels increased significantly (approx. 80%) because the swelling of both test hydrogels increased considerably. The aforementioned results indicated that the swelling behaviors and drug-release profiles of these test hydrogels are significantly different due to their distinct cross-linking structures.     

Sonochemically prepared BSA microspheres containing Gemcitabine,and their potential application in renal cancer therapeutics

This report demonstrates the formation and characterization of sonochemically prepared bovine serum albumin (BSA)–Gemzar (Gemcitabine) microspheres and shows their increased anticancer activity compared to pristine Gemzar. The amount of loaded Gemzar was determined by light absorption measurements. The BSA–Gemzar composite was analyzed and characterized by optical microscopy and scanning electron microscopy. The release kinetics of Gemzar from the proteinaceous microspheres was tested. The BSA–Gemzar composite was examined for its anticancer activity (in vitro) in renal cancer cells (RCC, 786-O cells) using [³H]thymidine incorporation assays. It was found that the influence of the Gemzar-loaded microspheres on the cancer cells was significantly greater than that of an equimolar concentration of pristine Gemzar.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

The release profiles and bioactivity of parathyroid hormone from poly(lactic-co-glycolic acid) microspheres

Poly(lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) or human parathyroid hormone (PTH)(1-34) were prepared using a double emulsion method with high encapsulation efficiency and controlled particle sizes. The microspheres were characterized with regard to their surface morphology, size, protein loading, degradation and release kinetics, and in vitro and in vivo assessments of biological activity of released PTH. PLGA5050 microspheres degraded rapidly after a 3-week lag time and were degraded completely within 4 months. In vitro BSA release kinetics from PLGA5050 microspheres were characterized by a burst effect followed by a slow release phase within 1-7 weeks and a second burst release at 8 weeks, which was consistent with the degradation study. The PTH incorporated PLGA5050 microspheres released detectable PTH in the initial 24h, and the released PTH was biologically active as evidenced by the stimulated release of cAMP from ROS 17/2.8 osteosarcoma cells as well as increased serum calcium levels when injected subcutaneously into mice. Both in vitro and in vivo assays demonstrated that the bioactivity of PTH was maintained largely during the fabrication of PLGA microspheres and upon release. These studies illustrate the feasibility of achieving local delivery of PTH to induce a biologically active response in bone by a microsphere encapsulation technique.     

Preparation and characterization of photoresponsive poly(methyl methacrylate) microspheres bearing phosphorylcholine-analogous and spirooxazine moieties

The photoresponsive copolymer microspheres [poly(MAIP-co-MMA)] were prepared from the emulsifier-free emulsion copolymerization of 2-[2-(methacryloyloxy) ethyldimethylammonio]-ethyl indolinonaphthooxazine phosphate (MAIP) and methyl methacrylate (MMA). From the kinetics of the copolymerization of MAIP and MMA, it was found that the initial rate of polymerization of MMA increased by the addition of a small amount of MAIP. From the X-ray photoelectron spectroscopy (XPS) measurements MAIP moiety was found to be located on the surface of a particle. The introduction of a MAIP moiety into poly(MMA) microspheres results in a decrease in the amount of bovine serum albumin (BSA) adsorbed. A photoresponsive adsorption of BSA on poly(MAIP-co-MMA) microspheres was observed with spirooxazine-merocyanine photoisomerization caused by UV irradiation.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissueengineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Hollow and degradable polyelectrolyte nanocapsules for protein drug delivery

Biodegradable hollow capsules encapsulating protein drugs were prepared via layer-by-layer assembly of water-soluble chitosan and dextran sulfate on protein-entrapping amino-functionalized silica particles and the subsequent removal of the silica. In order to enhance the encapsulated efficiency and decrease its burst release, we designed this new system to fulfill these two goals. Bovine serum albumin (BSA), which was used as model protein, was entrapped in the nanocapsules. This system demonstrated a good capacity for the encapsulation and loading of BSA. The burst release was decreased to less than 10% in phosphate-buffered saline within 2 h. No significant conformation change was noted from the released BSA in comparison with native BSA by using circular dichroism spectroscopy. Cell viability study suggested that the nanocapsules had good biocompatibility. The drug release kinetics mechanism is Fickian diffusion. These kinds of novel composite nanocapsules may offer a promising delivery system for water-soluble proteins and peptides.     

Chitin/PLGA blend microspheres as a biodegradable drug delivery system: a new delivery system for protein

Novel chitin/PLGAs and chitin/PLA based microspheres were developed for the delivery of protein. These biodegradable microspheres were prepared by polymers blending and wet phase-inversion methods. The parameters such as selected non-solvents, temperature of water and ratio of polylactide to polyglycolide were adjusted to improve thermodynamic compatibility of individual polymer (chitin and PLGAs or chitin/PLA), which affects the hydration and degradation properties of the blend microspheres. Triphasic pattern of drug release model is observed from the release of protein from the chitin/PLGAs and chitin/PLA microspheres: the initially fast release (the first phase), the following slow release (the second phase) and the second burst release (the third phase). Formulations of the blends, which are based on the balance among the hydration rate of the chitin phase and degradation of chitin/PLA and PLGA phase, can lead to a controllable release of bovine serum albumin (BSA). In conclusion, such a chitin/PLGA 50/50 microsphere is novel and interesting, and may be used as a protein delivery system.     

Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds

Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.     

Alginate Films as Macromolecular Imprinted Matrices

Macromolecularly imprinted polymers have been developed to mimic the non-covalent interactions driving molecular recognition in nature. The creation of an engineered antibody mimic would allow for the development of customizable films for biomolecular sensing. To demonstrate this principle, a cross-linked alginate film has been imprinted with bovine serum albumin (BSA) using aqueous biocompatible gelation methods. The imprinting efficiency of the synthesized films imprinted with BSA was determined and compared to the non-specific uptake of complementary proteins which were not imprinted in the alginate matrix. It was found that the recognition of the BSA using an alginate film was 6.4 mg/g polymer, which compares favorably to previously reported macromolecularly imprinted networks. The absorption of non-imprinted cationic proteins by the alginate matrix demonstrates that overcoming non-specific binding needs to be a focus of future work in order to successfully employ these materials towards biomolecular sensing within a physiological environment.     

Extended and sequential delivery of protein from injectable thermoresponsive hydrogels

Thermoresponsive hydrogels are attractive for their injectability and retention in tissue sites where they may serve as a mechanical support and as a scaffold to guide tissue remodeling. Our objective in this report was to develop a thermoresponsive, biodegradable hydrogel system that would be capable of protein release from two distinct reservoirs--one where protein was attached to the hydrogel backbone, and one where protein was loaded into biodegradable microparticles mixed into the network. Thermoresponsive hydrogels consisting of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and biodegradable methacrylate polylactide were synthesized along with modified copolymers incorporating 1 mol % protein-reactive methacryloxy N-hydroxysuccinimide (MANHS), hydrophilic acrylic acid (AAc), or both. In vitro bovine serum albumin (BSA) release was studied from hydrogels, poly(lactide-co-glycolide) microparticles, or microparticles mixed into the hydrogels. The synthesized copolymers were able to gel below 37°C and release protein in excess of 3 months. The presence of MANHS and AAc in the copolymers was associated with higher loaded protein retention during thermal transition (45% vs. 22%) and faster release (2 months), respectively. Microspheres entrapped in the hydrogel released protein in a delayed fashion relative to microspheres in saline. The combination of a protein-reactive hydrogel mixed with protein-loaded microspheres demonstrated a sequential release of specific BSA populations. Overall the described drug delivery system combines the advantages of injectability, degradability, extended release, and sequential release, which may be useful in tissue engineering applications.     

Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method

The surface and internal morphology, drug distribution and release kinetics at 22 degrees C of polyesters such as PCL (polycaprolactone) and PLGA (poly(DL-lactic-co-glycolic acid)) 65:35 microspheres containing BSA (bovine serum albumin) have been investigated in order to understand the relationship amongst morphology, drug distribution and in vitro release profiles and to develop controlled release devices for marine fishes in tropical area. CLSM (confocal laser scanning microscope) micrographs reveal that the polyvinylalcohol (PVA as an emulsifier) concentration in the external water phase strongly influences drug distribution within microspheres and release profiles. The presence of PVA in the internal water phase enhances the stabilization of inner water droplets against coalescence. This results in a more uniform drug distribution and a slower BSA release. Different oil-phase volumes and polymer concentrations yield different solvent exchange and precipitation mechanisms, which lead to different morphologies. A low oil-phase volume yields microspheres with a porous matrix and defective skin surface, which gives a high initial BSA burst as well as a fast release profile. Microspheres fabricated from a low polymer concentration have less defective skin surface, but with a less tortuous inner matrix which results in a more rapid BSA release. A higher BSA loading yields a larger concentration gradient between the emulsion droplet and the continuous water phase as well as between the microspheres and the in vitro medium. The former results in a lower encapsulation efficiency, whereas the latter yields a faster initial burst and a more rapid release profile. High stirring speed can reduce microsphere size, but decreases the yield of microspheres.     

Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to Sepharose beads or nitrocellulose paper strips

Two novel immunization methods designed for immunization with small quantities of antigen, immobilized on a solid matrix and without the use of adjuvant, are presented. The major test antigen used in order to evaluate these methods was bovine serum albumin (BSA). It was deposited in the spleen of mice and rabbits, either attached to Sepharose beads (Pharmacia Sepharose 4B) or to nitrocellulose (NC) paper strips (Millipore). BSA was attached to NC by direct application or by electroblotting after SDS-polyacrylamide gel electrophoresis. The antibody response in mouse and rabbit serum, after intrasplenic immunizations with various quantities of antigen, was analyzed in an ELISA standard procedure. In mice, an antibody response in serum was detected after three intrasplenic immunizations with a total quantity of 73.6 ng BSA bound to Sepharose beads and after two immunizations with a total quantity of 800 ng BSA attached to NC. Determination of the antigen-binding to NC and the clearance rate of antigen attached to NC deposited in the spleen of mice was performed with 125I-labeled BSA. In rabbits, an antibody response in serum was detected after a single intrasplenic immunization with 2.6 micrograms BSA attached to NC. When testing human insulin and sheep prolactin, attached to NC, as antigens in intrasplenic immunization of rabbits, an antibody response was found after a total quantity of 3.2 micrograms insulin and 10.5 micrograms prolactin, respectively.