Entrapment in plasma microparticles: A promising strategy for antigen delivery Munazza T. Fatima, Ejaj Ahmad, Mohammed Saleemuddin The Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 29 July 2013; revised 21 November 2013; accepted 4 January 2014 Published online 6 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33108 Abstract: We report the preparation of plasma microparticles (PMPs) from autologous blood plasma for sustained in vivo delivery of the entrapped antigens. The PMPs were prepared by high speed-stirring of calcium-enriched plasma, mixed with the antigen to be entrapped, in mineral oil. The preparation of PMPs did not necessitate addition of any external protein/enzyme nor special laboratory setup. Our results suggest that the PMPs release the entrapped invertase in a sustained manner both in vitro and in vivo, especially after crosslinking with glutaraldehyde. The preparations are reasonably stable to proteolysis and constitute strong candidates for eliciting immune response. Induction of humoral immune response by the PMP-entrapped invertase, as evident from the high antibody titers, was remarkable and comparable with that observed in animals receiving the antigen emulsified with Freund’s Complete Adjuvant. Isotypic analysis of antibodies
showed a Th1-biased immune response in animals administered uncrosslinked or crosslinked PMPs-entrapped invertase, especially after a booster dose. The analysis in animals of the group immunized with adjuvant-emulsified antigen suggested a combined Th1 and Th2 response. PMP-entrapment also caused high expression of surface markers (CD80 and CD86) on antigen presenting cells, as well as effector T-cells surface markers (CD41 and CD81) as revealed by FACS. The study suggests that PMPs offer remarkable promise as adjuvant-free and biocompatible vaccine delivery systems. C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl BioV
mater, 102B: 1244–1254, 2014.
Key Words: adjuvant-free immunization, fibrin matrix, vaccine delivery, cell-mediated immune response, IgG2a/IgG1 ratios
How to cite this article: Fatima MT, Ahmad E, Saleemuddin M. 2014. Entrapment in plasma microparticles: A promising strategy for antigen delivery. J Biomed Mater Res Part B 2014:102B:1244–1254.
INTRODUCTION
The interest in micro/nano particle-based antigen delivery systems has gained remarkable momentum in the last decades, in view of their promise in controllable and sustained release of the entrapped antigens, their targeting as well as adjuvant-free induction of immunity. The administered particle-entrapped antigens are released in vivo either by pulsatile release from the carrier or by its slow degradation, thereby overcoming the need of multiple boosters.1 While it is widely recognized that particle dimensions may be crucial for effective delivery and vaccine adjuvant action, efforts to correlate particle size with these actions have not been successful with claims of superiority both of micron and submicron-sized particles.2 Vaccines associated with several biodegradable systems are efficient generators of immune response against microbial, viral, and/or tumorigenic protein antigens. Studies with biodegradable polymers such as poly (L-lactide) (PLA), Poly (D, L-lactide-co-glycolide) (PLGA), or poly (ethylene glycol) (PEG) suggest their potential for the delivery of vaccines.3,4 Using nanoparticle-based vaccine delivery systems, it is possible to control release rate of the antigen, target to dendrite cells (DCs), and activate the anti-
gen presenting cells (APCs).1 The PLGA-entrapped antigens are targeted to the APCs and augment immune response.5–8 Chitosan-based microparticles and nanoparticles have also proved their potential as vaccine candidates.9–12 In spite of their established biodegradability, however, several synthetic micro/nanoparticle-based delivery systems suffer from problems of biocompatibility.13 Fibrin matrices, originally employed as sealants to prevent blood loss during surgery, are currently being explored as highly biocompatible and biodegradable systems for sustained delivery of a variety of antibiotics, therapeutic proteins/enzymes, peptides, genes, and even different types of cells.14–16 Thrombolytic cleavage of fibrinogen, yields fibrin, that rapidly undergoes polymerization to a threedimensional network and factor XIIIa (transglutaminase) confers stability to the network by creating covalent crosslinks.17 A number of fibrin-based preparations including gels,18 glues,19 clots,18 discs,20 films,21 fibers,22 microbeads,23 and nano particles,24 have been investigated for their drug delivery potential. Besides these, several fibrin composite scaffolds containing various stable synthetic and natural polymers have been prepared for the delivery of
Correspondence to: M. Saleemuddin (e-mail: [email protected])
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antibiotics, therapeutic proteins, and antigens.25–27 A simple procedure for the preparation of plasma clot in beaded form for the entrapment of small/large molecular weight drugs/proteins was developed in this laboratory. The beads could be conveniently prepared using autologous plasma, by activating endogenous thrombin and transglutaminase.28 More recently, the vaccine potential of C. albicans cytosolic proteins (Cp) entrapped in the plasma beads for immunization of BALB/c mice was investigated. The plasma beadentrapped Cp were highly immunogenic, induced both humoral and cell-mediated immunity and the response could be favorably altered by pre-entrapment of the antigen in liposomes.27 In this paper we report the immunogenic potential of the plasma microparticle (PMP)-entrapped model antigen, yeast invertase in rabbits and mice. Yeast invertase was chosen as a model antigen because of its high immunogenicity.29 MATERIALS AND METHODS
Chemicals Invertase, Amphotericin B, trypsin, chymotrypsin, bicinchoninic acid (BCA) total protein determination kit, Complete Freund’s Adjuvant (CFA) and Incomplete Freund’s Adjuvant (IFA) were purchased from Sigma Chemical Company, St. Louis, USA. Ethylene diamine tetra acetic acid (EDTA), CaCl2 and Tween 20 were obtained from SISCO Research Laboratory, Mumbai, India. Triton-X 100 was from Qualigens Fine Chemicals, Mumbai, India. Rabbit anti-mouse IgG-peroxidase conjugate and goat anti-rabbit IgG-HRP were purchased from Genei, India. Mouse Immunoglobulin isotyping ELISA Kit, monoclonal anti-mouse CD41, CD81, CD80, and CD86 and their isotypic controls were purchased from BD Biosciences, USA. Other chemicals used in study were of analytical grade. Animals, collection of blood and isolation of plasma/serum Inbred female Balb/c mice (6–8 weeks old), weighing 23–27 g were procured from the animal house facility of Central Drug Research Institute or CSIR- institute of Toxicological Research, Lucknow, India. Rabbits used in the study were out-bred and purchased from a local animal supplier. All the animals were provided with water and diet ad libitum (Hindustan Lever, India) throughout the investigation, unless mentioned otherwise. Bleeding, injections, and sacrifice of animals were strictly performed following the mandates approved by the Institutional Animal Ethics Committee constituted as per the recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Rabbit blood was collected from the ear vein with help of a syringe. Nine volumes of blood were mixed in tubes containing one volume of 2.7% (w/v) EDTA in normal saline, mixed and centrifuged at 2400 g for 10 min. Mice were bled through retro-orbital puncture with a glass capillary and blood collected in sterile Eppendorf tubes. For collection of serum, blood was incubated at room temperature for 1 h, kept at 4 C for 2 h for retraction of the clot and
centrifuged at 2400g for 10 min at 4 C. Both plasma and serum were collected by aspiration and stored if required at 220 C. Preparation of PMPs The procedure described earlier for obtaining the fibrin micro/nanoparticles in the oil system with high-speed stirring,30 with some modifications was followed. Briefly, PMPs were prepared using a set up comprising of a beaker containing paraffin oil kept in a larger water-filled beaker and placed on a thermostatic hot plate. A high speed mechanical stirrer was employed to stir the oil. One milliliter of plasma was taken in a microfuge tube and mixed with 100 lL of the antigen to be entrapped dissolved in PBS. Adequate CaCl2 was added to achieve a final concentration of 40 mM. The mixture was immediately transferred to a syringe with 23G hi-tech needle and dropped into a beaker containing paraffin oil stirred at 8000 rpm. The contents of the beaker were stirred for various durations (30–75 min) and temperatures (40–50 C). The PMPs formed were sedimented by centrifugation at 10,000g for 10 min, washed twice with diethyl ether and thrice with a mixture of n-hexane and isopropanol in a ratio of 3:1 to remove the oil phase.31 Where indicated the PMPs were treated with 0.05% glutaraldehyde for 15 min at 4 C, washed with the buffer repeatedly with the final wash containing 0.1M methylamine. The particle size was measured using NANOPHOX Particle Size Analyzer with Photon CROSS Correlation Spectroscopy (PCCS) (Sympatec, Germany) using WINDOX 5 software. Sample preparation for scanning electron microscopy (SEM) The PMPs were placed at 37 C to evaporate all organic solvent and suspended in 0.1M phosphate buffer, pH 7.4 containing 2.5% (v/v) glutaraldehyde for 2 h at room temperature. They were washed with the same buffer to remove excess glutaraldehyde, mounted over the cover slips and air dried. The PMPs were again washed with distilled water and successively with a gradient of ethanol water mixture (50%, 70%, 90%, and 100%). Finally the particles were dried and coated with platinum–palladium for electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) For the analysis of the polypeptide composition, the PMPs were dissolved in 0.1N NaOH and protein was estimated by BCA method. Further, same amount of PMPs were dissolved in 500 mL of 5 x sample loading buffer containing 50% glycerol, 10% (w/v) sodium dodecyl sulfate (SDS), 1.0M Tris-HCl pH 6.8, 40 mM b-mercaptoethanol and 1% (w/v) bromophenol blue. Aliquots were boiled in a water bath and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as described by Laemmlli (1970).32 Release of proteins from PMPs In vitro release of Invertase-loaded in PMPs was quantitated by suspending the PMPs containing entrapped enzyme (250
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mg Invertase/7 mg PMPs) in 1.5 mL of 20 mM phosphate buffered saline. The supernatant (20 mL) was periodically collected by centrifuging the PMPs at 10, 000g. The enzyme release was quantitated with the help of a standard curve. The total enzyme content was calculated by dissolving the PMPs in 1% Triton X-100. Where the PMPs (loaded with protein/drug) indicated was additionally treated with 0.05% (v/v) glutaraldehyde in 1.0 mL PBS at 4 C for 15 min to facilitate crosslinking. Crosslinking was stopped by adding 500 mL of 0.5M glycine or methylamine, and the PMPs washed thrice with normal saline and finally in PBS to follow the release kinetics or to perform in vivo experiments. Proteolysis of PMPs and crosslinked PMPs in the presence of trypsin and chymotrypsin In a total volume of 1.0 mL PBS, PMPs (7 mg protein) prepared with or without glutaraldehyde treatment were incubated with trypsin (20 mg) and chymotrypsin. The samples were incubated at 25 C, centrifuged at 10,000g and aliquots of the supernatant analyzed for protein by the BCA procedure. Uncrosslinked and crosslinked PMPs, incubated under the conditions without proteases constituted the respective controls. For calculation of extent of proteolysis the protein concentration of untreated PMPs, after dissolving in 1% triton was taken as 100. Immunization of rabbits and mice with PMP-entrapped invertase Three rabbits weighing 1.3, 1.5, and 1.7 kg were used for the study. Animals in the group I were subcutaneously administered CFA-Inv while those in groups II and III received intraperitoneally, invertase-entrapped uncrosslinked PMPs (PMP-Inv) or those crosslinked (0.05% for 15 min) (PMP-Inv-CL), respectively. Each animal received 600 mg antigens. A booster dose of 200 mg was given through the same routes on day 35 after the primary immunization. In case of the animals receiving the adjuvants, however, the booster dose was given with after emulsification of invertase with Freund’s incomplete adjuvant (IFA). Inbred female Balb/c mice were used. Four groups of mice each comprising eight animals weighing approximately 25 g (maximum 27g, minimum 23g) were used. The animals received invertase dissolved in saline, (Sal-Inv), emulsified with CFA, (CFA-Inv), invertase-entrapped in PMPs (PMP-Inv) and enzyme invertase-entrapped and glutaraldehyde crosslinked PMPs (PMP-Inv- CL). Invertase dissolved in saline and PMPentrapped antigen was administered i.p., whereas CFA-Inv group received the antigen subcutaneously. The animals received 60 mg of the antigen in various formulations as the priming dose and boosters contained 20 mg of invertase on day 21. Sera were collected at regular intervals and indirect ELISA33 performed to analyze antibody levels. The antibody isotypes were quantitated using procedure described in the Mouse Immunoglobulin Isotyping ELISA Kit (BD Biosciences). Staining for T cell and co-stimulatory surface markers On day 5 postadministration of the booster, three mice from each group were sacrificed by cervical dislocation. Spleens
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were aseptically removed, washed with PBS and then crushed with the rough end of the frosted end slides. The resulting suspensions were taken in the culture tubes and centrifuged at 4 C at 200g for 8 min and supernatant was discarded. The pellet was then suspended in ACK lysis buffer (10–15 mL) for 15 min for the lysis of the erythrocytes. The splenocytes were washed with RPMI 1640 medium containing 10% fetal calf serum. Viable cells were counted by 0.1% Trypan Blue dye exclusion procedure. Various surface markers present on the APS were also studied. The splenocytes were harvested as described earlier for cell proliferation assay, 1x106 cells were taken into microfuge tubes containing 1.0 mL FACS buffer (PBS with 1% BSA and 0.1% sodium azide), washed twice with the same buffer and centrifuged at 200g for 10 min. To the cells was added the Fc block (2.4 G2), followed by 125 ng of FITC/PE tagged monoclonal antibodies (CD4, CD8, CD80, CD86 or anti-isotype control). The tubes were incubated at 4 C for 30 min, the splenocytes washed twice with the FACS buffer to remove unbound antibodies and fixed with 1% (v/v) paraformaldehyde. The flow cytometry data were obtained using a fluorescence activated cell sorter (GUAVA, Billerica, MA) and data analyzed using the express plus software (GUAVA, USA). Statistical analysis All the data were analyzed by one-way analysis of variance (ANOVA) following Holm-Sidak or Tukey test method (All pairwise multiple comparison methods) and Dunnett test (comparison vs control). Values of p < 0.05 were considered statistically significant. RESULTS
Characterization of the PMPs Particle-size analysis (Figure 1) suggested that the PMPs prepared at 40 C have diameters in the range of 1.0–5.0 mm, with majority lying between 2.0 and 3.0 mm. As shown in the insert, the particles appeared nearly spherical with rather smooth surface in SEM and entrapment of inveratse caused no difference in the shape or dimensions of the particles. SDS-PAGE of the various PMP formulations suggested a polypeptide composition comparable to that of plasma, but increase in time of stirred incubation resulted in increase in the bands in the region above 70 kDa (Figure 2). The PMPs prepared without added calcium showed fewer of the high molecular weight bands. SDS-PAGE of PMPs incubated up to 50 C or for longer duration did not increase the high molecular weight bands (data not included). Invertase added to the calcium-enriched plasma prior to the stirring in mineral oil was effectively entrapped in the PMPs. The amount of entrapped invertase increased with the length of the stirred incubation but reached a plateau at 60 min [Figure 3(A)]. Entrapment yields of invertase were remarkably high with almost all the added enzyme retained in the PMPs when the stirring was continued at 40 C for 60 min or beyond [Figure 3(B)]. Further increase in incubation time and/or temperature did not improve the entrapment yield of the enzyme. Enzyme entrapment in PMPs prepared
PLASMA MICROPARTICLE-BASED ANTIGEN DELIVERY
ORIGINAL RESEARCH REPORT
FIGURE 1. Particle size of PMPs. Particle size was measured using NANOPHOX Particle Size Analyzer employing Photon CROSS Correlation Spectroscopy (x10 5 1356.55 nm, x905 2188.01 nm, SMD (Standard mean deviation) 5 1674.38 nm, VMD (Volume mean diameter) 5 1748.86 nm, N 5 3). Insert shows the SEM images of PMPs without (Panel A) or entrapped invertase (Panel B). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
without added calcium was, however, very low. Most of the experiments described in this study to produce the PMPs were performed using the preparations obtained by stirring plasma in the presence of 40 mM CaCl2 for 75 min.
Proteolysis of the PMPs Proteolysis of the uncrosslinked PMPs and those treated with glutaraldehyde was undertaken with a view to study the possible in vivo stability (Figure 5). Release of proteins
Release of invertase from PMPs Figure 4 shows the in vitro release profile of invertase both from uncrosslinked or glutaraldehyde-treated PMPs. As evident from the figure, 26% of the entrapped invertase leaked into the medium from the PMPs in 72 h. Crosslinking with 0.05% (v/v) glutaraldehyde resulted in a further 50% decrease in the release of the enzyme (only 13% of invertase leaked out in 72 h).
FIGURE 2. SDS-PAGE of PMPs prepared with varying stirredincubation durations. Lane 1 contained molecular weight markers, while lane 2 was loaded with PMPs prepared without added CaCl2. PMPs prepared with plasma containing 40 mM CaCl2 and incubated for 30, 45, and 60 min were loaded in Lanes 3, 4, and 5, respectively. The amount of protein loaded in each lane (2–5) is about 30 mg. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 3. Entrapment of Invertase in PMPs by varying incubation time at 40 C. PMPs were prepared as described in the text with or without added CaCl2. Entrapment of invertase (Panel A) and percent entrapment of added to invertase (Panel B) are shown. Each value represents the mean 6 SD of three determinations.
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FIGURE 4. Release of entrapped invertase from PMPs. PMPs containing entrapped invertase (36 mg/mg protein) were incubated in 1.5 mL PBS at 25 C and the enzyme release was determined by assaying the activity as described under Methods. Invertase release was investigated from PMPs prepared at 40 C (‡), prepared at 40 C in presence of 0.05% glutaraldehyde (䉬) or prepared at 50 C (~). Each value represents 6 SD of four determinations.
from the PMPs incubated with trypsin (20 mg/mL) or chymotrypsin (20 mg/mL) was monitored up to 72 h. The PMPs incubated without protease and those incubated with trypsin and chymotrypsin released 17%, 24%, and 31% of the total proteins into the medium, respectively, after an initial burst observed at 2 h. Crosslinked PMPs not exposed to the enzyme or those treated with trypsin and chymotrypsin released only 10%, 12%, and 14% of the total protein, respectively, under the conditions. The results suggest that PMPs are stable to proteolysis by more than one enzyme at neutral pH, especially after glutaraldehyde treatment. Immune response in rabbits against invertase-entrapped PMPs Immunization potential of PMP-entrapped invertase was also investigated. Figure 6(A) shows the anti-invertase antibody response in rabbits after immunization with invertase emulsified with CFA (CFA-Inv), PMP-entrapped invertase (PMP-Inv) and the PMP-Inv crosslinked with glutaraldehyde (PMP-Inv-CL). All the animals receiving the antigen through various formulations showed increase in the level of antiinvertase IgG in sera till day 20 postimmunizations. Highest titers were recorded in animals that received CFA-Inv followed by PMP-Inv and PMP-Inv-CL formulations. The titers however decreased gradually by day 35 in all the groups of animals with the exception of those that received the PMPInv-CL. In this group the IgG titer increased till the day 28 and showed only a small decrease on day 35. The antibody titers in all the three groups were however comparable by day 28. Administration of the booster dose resulted in enhancement of antibody titers in all groups of animals also in the order; PMP-Inv-CL> IFA-Inv PMP-Inv [Figure 6(B)]. Immune response in mice against the invertase-entrapped PMPs Immunization of mice with PMP-entrapped invertase gave response qualitatively comparable that observed in rabbits,
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both postprimary immunization and booster dose but the titers of antibodies were lower in the former [Figure 7(A,B)]. The antibody level in the PMP-Inv-CL group was moderate at 14 days after immunization, but the levels increased beyond those of the adjuvant and uncrosslinked preparation administered animals by day 35 of administration. Animals receiving PMP-Inv-CL also exhibited superior antibody titer after administration of the booster dose as compared to those receiving the adjuvant as well as the uncrosslinked preparations. Antibody levels in the groups of animals receiving uncrosslinked preparations, both on day 10 and day 20 after the administration of the booster, were comparable with those receiving the antigen emulsified with the CFA-Inv. Invertase administered without adjuvant or entrapment (Sal-Inv) was only weakly antigenic both after the primary immunization and the booster dose. IgG2a/IgG1 ratios in mice receiving PMP-entrapped invertase Mean IgG2a/IgG1 isotype ratio in all the mice receiving invertase emulsified with CFA or entrapped in crosslinked or uncrosslinked PMPs was approximately 1 (0.98–1.04), 20 days after the administration of the first dose of the antigen
FIGURE 5. In vitro proteolysis of PMPs. Various preparations of PMPs (containing 7 mg protein) were incubated in a total volume of 1.0 mL with 20 mg/mL trypsin (A) or 20 mg/mL of chymotrypsin (B). Preparations investigated include: uncross-linked (䊏) and cross-linked PMPs (•). Solubilization of protein from uncross-linked (w) and cross-linked (j) PMPs not exposed to the protease was also studied. All samples were incubated in phosphate buffered saline at 25 C and solubilized protein release was quantitated by BCA as described under Methods. Each value represents mean 6 SD of four determinations.
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ORIGINAL RESEARCH REPORT
FIGURE 6. Antibody response in rabbit after primary immunization (Panel A) and postbooster (Panel B) with PMPs containing entrapped invertase. Groups of rabbits were immunized with various invertase formulations including the enzyme emulsified in CFA (CFA-Inv), entrapped in uncrosslinked PMPs (PMP-Inv) and cross-linked PMPs (PMP-Inv-CL) as described under methods. Sera were collected and ELISA performed to quantitate total IgG on days 10, 20, 28, and 35 after the primary immunization. All animals received the formulations containing 600 mg of invertase as a single dose. Values represent the mean 6 SD of three determinations at 1:800 dilutions of the sera. On the day 35 postimmunization, animal were given booster dose with respective formulations containing 20 mg invertase. The antibody titer was analyzed on day 12 and day 24. Values indicate the mean 6 SD of three determinants at 1:3200 dilutions of the sera. P values among the various statistically significant groups on day 20 (PMP-Inv vs PMP-Inv-CL
showed a Th1-biased immune response in animals administered uncrosslinked or crosslinked PMPs-entrapped invertase, especially after a booster dose. The analysis in animals of the group immunized with adjuvant-emulsified antigen suggested a combined Th1 and Th2 response. PMP-entrapment also caused high expression of surface markers (CD80 and CD86) on antigen presenting cells, as well as effector T-cells surface markers (CD41 and CD81) as revealed by FACS. The study suggests that PMPs offer remarkable promise as adjuvant-free and biocompatible vaccine delivery systems. C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl BioV
mater, 102B: 1244–1254, 2014.
Key Words: adjuvant-free immunization, fibrin matrix, vaccine delivery, cell-mediated immune response, IgG2a/IgG1 ratios
How to cite this article: Fatima MT, Ahmad E, Saleemuddin M. 2014. Entrapment in plasma microparticles: A promising strategy for antigen delivery. J Biomed Mater Res Part B 2014:102B:1244–1254.
INTRODUCTION
The interest in micro/nano particle-based antigen delivery systems has gained remarkable momentum in the last decades, in view of their promise in controllable and sustained release of the entrapped antigens, their targeting as well as adjuvant-free induction of immunity. The administered particle-entrapped antigens are released in vivo either by pulsatile release from the carrier or by its slow degradation, thereby overcoming the need of multiple boosters.1 While it is widely recognized that particle dimensions may be crucial for effective delivery and vaccine adjuvant action, efforts to correlate particle size with these actions have not been successful with claims of superiority both of micron and submicron-sized particles.2 Vaccines associated with several biodegradable systems are efficient generators of immune response against microbial, viral, and/or tumorigenic protein antigens. Studies with biodegradable polymers such as poly (L-lactide) (PLA), Poly (D, L-lactide-co-glycolide) (PLGA), or poly (ethylene glycol) (PEG) suggest their potential for the delivery of vaccines.3,4 Using nanoparticle-based vaccine delivery systems, it is possible to control release rate of the antigen, target to dendrite cells (DCs), and activate the anti-
gen presenting cells (APCs).1 The PLGA-entrapped antigens are targeted to the APCs and augment immune response.5–8 Chitosan-based microparticles and nanoparticles have also proved their potential as vaccine candidates.9–12 In spite of their established biodegradability, however, several synthetic micro/nanoparticle-based delivery systems suffer from problems of biocompatibility.13 Fibrin matrices, originally employed as sealants to prevent blood loss during surgery, are currently being explored as highly biocompatible and biodegradable systems for sustained delivery of a variety of antibiotics, therapeutic proteins/enzymes, peptides, genes, and even different types of cells.14–16 Thrombolytic cleavage of fibrinogen, yields fibrin, that rapidly undergoes polymerization to a threedimensional network and factor XIIIa (transglutaminase) confers stability to the network by creating covalent crosslinks.17 A number of fibrin-based preparations including gels,18 glues,19 clots,18 discs,20 films,21 fibers,22 microbeads,23 and nano particles,24 have been investigated for their drug delivery potential. Besides these, several fibrin composite scaffolds containing various stable synthetic and natural polymers have been prepared for the delivery of
Correspondence to: M. Saleemuddin (e-mail: [email protected])
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ORIGINAL RESEARCH REPORT
antibiotics, therapeutic proteins, and antigens.25–27 A simple procedure for the preparation of plasma clot in beaded form for the entrapment of small/large molecular weight drugs/proteins was developed in this laboratory. The beads could be conveniently prepared using autologous plasma, by activating endogenous thrombin and transglutaminase.28 More recently, the vaccine potential of C. albicans cytosolic proteins (Cp) entrapped in the plasma beads for immunization of BALB/c mice was investigated. The plasma beadentrapped Cp were highly immunogenic, induced both humoral and cell-mediated immunity and the response could be favorably altered by pre-entrapment of the antigen in liposomes.27 In this paper we report the immunogenic potential of the plasma microparticle (PMP)-entrapped model antigen, yeast invertase in rabbits and mice. Yeast invertase was chosen as a model antigen because of its high immunogenicity.29 MATERIALS AND METHODS
Chemicals Invertase, Amphotericin B, trypsin, chymotrypsin, bicinchoninic acid (BCA) total protein determination kit, Complete Freund’s Adjuvant (CFA) and Incomplete Freund’s Adjuvant (IFA) were purchased from Sigma Chemical Company, St. Louis, USA. Ethylene diamine tetra acetic acid (EDTA), CaCl2 and Tween 20 were obtained from SISCO Research Laboratory, Mumbai, India. Triton-X 100 was from Qualigens Fine Chemicals, Mumbai, India. Rabbit anti-mouse IgG-peroxidase conjugate and goat anti-rabbit IgG-HRP were purchased from Genei, India. Mouse Immunoglobulin isotyping ELISA Kit, monoclonal anti-mouse CD41, CD81, CD80, and CD86 and their isotypic controls were purchased from BD Biosciences, USA. Other chemicals used in study were of analytical grade. Animals, collection of blood and isolation of plasma/serum Inbred female Balb/c mice (6–8 weeks old), weighing 23–27 g were procured from the animal house facility of Central Drug Research Institute or CSIR- institute of Toxicological Research, Lucknow, India. Rabbits used in the study were out-bred and purchased from a local animal supplier. All the animals were provided with water and diet ad libitum (Hindustan Lever, India) throughout the investigation, unless mentioned otherwise. Bleeding, injections, and sacrifice of animals were strictly performed following the mandates approved by the Institutional Animal Ethics Committee constituted as per the recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Rabbit blood was collected from the ear vein with help of a syringe. Nine volumes of blood were mixed in tubes containing one volume of 2.7% (w/v) EDTA in normal saline, mixed and centrifuged at 2400 g for 10 min. Mice were bled through retro-orbital puncture with a glass capillary and blood collected in sterile Eppendorf tubes. For collection of serum, blood was incubated at room temperature for 1 h, kept at 4 C for 2 h for retraction of the clot and
centrifuged at 2400g for 10 min at 4 C. Both plasma and serum were collected by aspiration and stored if required at 220 C. Preparation of PMPs The procedure described earlier for obtaining the fibrin micro/nanoparticles in the oil system with high-speed stirring,30 with some modifications was followed. Briefly, PMPs were prepared using a set up comprising of a beaker containing paraffin oil kept in a larger water-filled beaker and placed on a thermostatic hot plate. A high speed mechanical stirrer was employed to stir the oil. One milliliter of plasma was taken in a microfuge tube and mixed with 100 lL of the antigen to be entrapped dissolved in PBS. Adequate CaCl2 was added to achieve a final concentration of 40 mM. The mixture was immediately transferred to a syringe with 23G hi-tech needle and dropped into a beaker containing paraffin oil stirred at 8000 rpm. The contents of the beaker were stirred for various durations (30–75 min) and temperatures (40–50 C). The PMPs formed were sedimented by centrifugation at 10,000g for 10 min, washed twice with diethyl ether and thrice with a mixture of n-hexane and isopropanol in a ratio of 3:1 to remove the oil phase.31 Where indicated the PMPs were treated with 0.05% glutaraldehyde for 15 min at 4 C, washed with the buffer repeatedly with the final wash containing 0.1M methylamine. The particle size was measured using NANOPHOX Particle Size Analyzer with Photon CROSS Correlation Spectroscopy (PCCS) (Sympatec, Germany) using WINDOX 5 software. Sample preparation for scanning electron microscopy (SEM) The PMPs were placed at 37 C to evaporate all organic solvent and suspended in 0.1M phosphate buffer, pH 7.4 containing 2.5% (v/v) glutaraldehyde for 2 h at room temperature. They were washed with the same buffer to remove excess glutaraldehyde, mounted over the cover slips and air dried. The PMPs were again washed with distilled water and successively with a gradient of ethanol water mixture (50%, 70%, 90%, and 100%). Finally the particles were dried and coated with platinum–palladium for electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) For the analysis of the polypeptide composition, the PMPs were dissolved in 0.1N NaOH and protein was estimated by BCA method. Further, same amount of PMPs were dissolved in 500 mL of 5 x sample loading buffer containing 50% glycerol, 10% (w/v) sodium dodecyl sulfate (SDS), 1.0M Tris-HCl pH 6.8, 40 mM b-mercaptoethanol and 1% (w/v) bromophenol blue. Aliquots were boiled in a water bath and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) as described by Laemmlli (1970).32 Release of proteins from PMPs In vitro release of Invertase-loaded in PMPs was quantitated by suspending the PMPs containing entrapped enzyme (250
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mg Invertase/7 mg PMPs) in 1.5 mL of 20 mM phosphate buffered saline. The supernatant (20 mL) was periodically collected by centrifuging the PMPs at 10, 000g. The enzyme release was quantitated with the help of a standard curve. The total enzyme content was calculated by dissolving the PMPs in 1% Triton X-100. Where the PMPs (loaded with protein/drug) indicated was additionally treated with 0.05% (v/v) glutaraldehyde in 1.0 mL PBS at 4 C for 15 min to facilitate crosslinking. Crosslinking was stopped by adding 500 mL of 0.5M glycine or methylamine, and the PMPs washed thrice with normal saline and finally in PBS to follow the release kinetics or to perform in vivo experiments. Proteolysis of PMPs and crosslinked PMPs in the presence of trypsin and chymotrypsin In a total volume of 1.0 mL PBS, PMPs (7 mg protein) prepared with or without glutaraldehyde treatment were incubated with trypsin (20 mg) and chymotrypsin. The samples were incubated at 25 C, centrifuged at 10,000g and aliquots of the supernatant analyzed for protein by the BCA procedure. Uncrosslinked and crosslinked PMPs, incubated under the conditions without proteases constituted the respective controls. For calculation of extent of proteolysis the protein concentration of untreated PMPs, after dissolving in 1% triton was taken as 100. Immunization of rabbits and mice with PMP-entrapped invertase Three rabbits weighing 1.3, 1.5, and 1.7 kg were used for the study. Animals in the group I were subcutaneously administered CFA-Inv while those in groups II and III received intraperitoneally, invertase-entrapped uncrosslinked PMPs (PMP-Inv) or those crosslinked (0.05% for 15 min) (PMP-Inv-CL), respectively. Each animal received 600 mg antigens. A booster dose of 200 mg was given through the same routes on day 35 after the primary immunization. In case of the animals receiving the adjuvants, however, the booster dose was given with after emulsification of invertase with Freund’s incomplete adjuvant (IFA). Inbred female Balb/c mice were used. Four groups of mice each comprising eight animals weighing approximately 25 g (maximum 27g, minimum 23g) were used. The animals received invertase dissolved in saline, (Sal-Inv), emulsified with CFA, (CFA-Inv), invertase-entrapped in PMPs (PMP-Inv) and enzyme invertase-entrapped and glutaraldehyde crosslinked PMPs (PMP-Inv- CL). Invertase dissolved in saline and PMPentrapped antigen was administered i.p., whereas CFA-Inv group received the antigen subcutaneously. The animals received 60 mg of the antigen in various formulations as the priming dose and boosters contained 20 mg of invertase on day 21. Sera were collected at regular intervals and indirect ELISA33 performed to analyze antibody levels. The antibody isotypes were quantitated using procedure described in the Mouse Immunoglobulin Isotyping ELISA Kit (BD Biosciences). Staining for T cell and co-stimulatory surface markers On day 5 postadministration of the booster, three mice from each group were sacrificed by cervical dislocation. Spleens
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were aseptically removed, washed with PBS and then crushed with the rough end of the frosted end slides. The resulting suspensions were taken in the culture tubes and centrifuged at 4 C at 200g for 8 min and supernatant was discarded. The pellet was then suspended in ACK lysis buffer (10–15 mL) for 15 min for the lysis of the erythrocytes. The splenocytes were washed with RPMI 1640 medium containing 10% fetal calf serum. Viable cells were counted by 0.1% Trypan Blue dye exclusion procedure. Various surface markers present on the APS were also studied. The splenocytes were harvested as described earlier for cell proliferation assay, 1x106 cells were taken into microfuge tubes containing 1.0 mL FACS buffer (PBS with 1% BSA and 0.1% sodium azide), washed twice with the same buffer and centrifuged at 200g for 10 min. To the cells was added the Fc block (2.4 G2), followed by 125 ng of FITC/PE tagged monoclonal antibodies (CD4, CD8, CD80, CD86 or anti-isotype control). The tubes were incubated at 4 C for 30 min, the splenocytes washed twice with the FACS buffer to remove unbound antibodies and fixed with 1% (v/v) paraformaldehyde. The flow cytometry data were obtained using a fluorescence activated cell sorter (GUAVA, Billerica, MA) and data analyzed using the express plus software (GUAVA, USA). Statistical analysis All the data were analyzed by one-way analysis of variance (ANOVA) following Holm-Sidak or Tukey test method (All pairwise multiple comparison methods) and Dunnett test (comparison vs control). Values of p < 0.05 were considered statistically significant. RESULTS
Characterization of the PMPs Particle-size analysis (Figure 1) suggested that the PMPs prepared at 40 C have diameters in the range of 1.0–5.0 mm, with majority lying between 2.0 and 3.0 mm. As shown in the insert, the particles appeared nearly spherical with rather smooth surface in SEM and entrapment of inveratse caused no difference in the shape or dimensions of the particles. SDS-PAGE of the various PMP formulations suggested a polypeptide composition comparable to that of plasma, but increase in time of stirred incubation resulted in increase in the bands in the region above 70 kDa (Figure 2). The PMPs prepared without added calcium showed fewer of the high molecular weight bands. SDS-PAGE of PMPs incubated up to 50 C or for longer duration did not increase the high molecular weight bands (data not included). Invertase added to the calcium-enriched plasma prior to the stirring in mineral oil was effectively entrapped in the PMPs. The amount of entrapped invertase increased with the length of the stirred incubation but reached a plateau at 60 min [Figure 3(A)]. Entrapment yields of invertase were remarkably high with almost all the added enzyme retained in the PMPs when the stirring was continued at 40 C for 60 min or beyond [Figure 3(B)]. Further increase in incubation time and/or temperature did not improve the entrapment yield of the enzyme. Enzyme entrapment in PMPs prepared
PLASMA MICROPARTICLE-BASED ANTIGEN DELIVERY
ORIGINAL RESEARCH REPORT
FIGURE 1. Particle size of PMPs. Particle size was measured using NANOPHOX Particle Size Analyzer employing Photon CROSS Correlation Spectroscopy (x10 5 1356.55 nm, x905 2188.01 nm, SMD (Standard mean deviation) 5 1674.38 nm, VMD (Volume mean diameter) 5 1748.86 nm, N 5 3). Insert shows the SEM images of PMPs without (Panel A) or entrapped invertase (Panel B). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
without added calcium was, however, very low. Most of the experiments described in this study to produce the PMPs were performed using the preparations obtained by stirring plasma in the presence of 40 mM CaCl2 for 75 min.
Proteolysis of the PMPs Proteolysis of the uncrosslinked PMPs and those treated with glutaraldehyde was undertaken with a view to study the possible in vivo stability (Figure 5). Release of proteins
Release of invertase from PMPs Figure 4 shows the in vitro release profile of invertase both from uncrosslinked or glutaraldehyde-treated PMPs. As evident from the figure, 26% of the entrapped invertase leaked into the medium from the PMPs in 72 h. Crosslinking with 0.05% (v/v) glutaraldehyde resulted in a further 50% decrease in the release of the enzyme (only 13% of invertase leaked out in 72 h).
FIGURE 2. SDS-PAGE of PMPs prepared with varying stirredincubation durations. Lane 1 contained molecular weight markers, while lane 2 was loaded with PMPs prepared without added CaCl2. PMPs prepared with plasma containing 40 mM CaCl2 and incubated for 30, 45, and 60 min were loaded in Lanes 3, 4, and 5, respectively. The amount of protein loaded in each lane (2–5) is about 30 mg. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 3. Entrapment of Invertase in PMPs by varying incubation time at 40 C. PMPs were prepared as described in the text with or without added CaCl2. Entrapment of invertase (Panel A) and percent entrapment of added to invertase (Panel B) are shown. Each value represents the mean 6 SD of three determinations.
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FIGURE 4. Release of entrapped invertase from PMPs. PMPs containing entrapped invertase (36 mg/mg protein) were incubated in 1.5 mL PBS at 25 C and the enzyme release was determined by assaying the activity as described under Methods. Invertase release was investigated from PMPs prepared at 40 C (‡), prepared at 40 C in presence of 0.05% glutaraldehyde (䉬) or prepared at 50 C (~). Each value represents 6 SD of four determinations.
from the PMPs incubated with trypsin (20 mg/mL) or chymotrypsin (20 mg/mL) was monitored up to 72 h. The PMPs incubated without protease and those incubated with trypsin and chymotrypsin released 17%, 24%, and 31% of the total proteins into the medium, respectively, after an initial burst observed at 2 h. Crosslinked PMPs not exposed to the enzyme or those treated with trypsin and chymotrypsin released only 10%, 12%, and 14% of the total protein, respectively, under the conditions. The results suggest that PMPs are stable to proteolysis by more than one enzyme at neutral pH, especially after glutaraldehyde treatment. Immune response in rabbits against invertase-entrapped PMPs Immunization potential of PMP-entrapped invertase was also investigated. Figure 6(A) shows the anti-invertase antibody response in rabbits after immunization with invertase emulsified with CFA (CFA-Inv), PMP-entrapped invertase (PMP-Inv) and the PMP-Inv crosslinked with glutaraldehyde (PMP-Inv-CL). All the animals receiving the antigen through various formulations showed increase in the level of antiinvertase IgG in sera till day 20 postimmunizations. Highest titers were recorded in animals that received CFA-Inv followed by PMP-Inv and PMP-Inv-CL formulations. The titers however decreased gradually by day 35 in all the groups of animals with the exception of those that received the PMPInv-CL. In this group the IgG titer increased till the day 28 and showed only a small decrease on day 35. The antibody titers in all the three groups were however comparable by day 28. Administration of the booster dose resulted in enhancement of antibody titers in all groups of animals also in the order; PMP-Inv-CL> IFA-Inv PMP-Inv [Figure 6(B)]. Immune response in mice against the invertase-entrapped PMPs Immunization of mice with PMP-entrapped invertase gave response qualitatively comparable that observed in rabbits,
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both postprimary immunization and booster dose but the titers of antibodies were lower in the former [Figure 7(A,B)]. The antibody level in the PMP-Inv-CL group was moderate at 14 days after immunization, but the levels increased beyond those of the adjuvant and uncrosslinked preparation administered animals by day 35 of administration. Animals receiving PMP-Inv-CL also exhibited superior antibody titer after administration of the booster dose as compared to those receiving the adjuvant as well as the uncrosslinked preparations. Antibody levels in the groups of animals receiving uncrosslinked preparations, both on day 10 and day 20 after the administration of the booster, were comparable with those receiving the antigen emulsified with the CFA-Inv. Invertase administered without adjuvant or entrapment (Sal-Inv) was only weakly antigenic both after the primary immunization and the booster dose. IgG2a/IgG1 ratios in mice receiving PMP-entrapped invertase Mean IgG2a/IgG1 isotype ratio in all the mice receiving invertase emulsified with CFA or entrapped in crosslinked or uncrosslinked PMPs was approximately 1 (0.98–1.04), 20 days after the administration of the first dose of the antigen
FIGURE 5. In vitro proteolysis of PMPs. Various preparations of PMPs (containing 7 mg protein) were incubated in a total volume of 1.0 mL with 20 mg/mL trypsin (A) or 20 mg/mL of chymotrypsin (B). Preparations investigated include: uncross-linked (䊏) and cross-linked PMPs (•). Solubilization of protein from uncross-linked (w) and cross-linked (j) PMPs not exposed to the protease was also studied. All samples were incubated in phosphate buffered saline at 25 C and solubilized protein release was quantitated by BCA as described under Methods. Each value represents mean 6 SD of four determinations.
PLASMA MICROPARTICLE-BASED ANTIGEN DELIVERY
ORIGINAL RESEARCH REPORT
FIGURE 6. Antibody response in rabbit after primary immunization (Panel A) and postbooster (Panel B) with PMPs containing entrapped invertase. Groups of rabbits were immunized with various invertase formulations including the enzyme emulsified in CFA (CFA-Inv), entrapped in uncrosslinked PMPs (PMP-Inv) and cross-linked PMPs (PMP-Inv-CL) as described under methods. Sera were collected and ELISA performed to quantitate total IgG on days 10, 20, 28, and 35 after the primary immunization. All animals received the formulations containing 600 mg of invertase as a single dose. Values represent the mean 6 SD of three determinations at 1:800 dilutions of the sera. On the day 35 postimmunization, animal were given booster dose with respective formulations containing 20 mg invertase. The antibody titer was analyzed on day 12 and day 24. Values indicate the mean 6 SD of three determinants at 1:3200 dilutions of the sera. P values among the various statistically significant groups on day 20 (PMP-Inv vs PMP-Inv-CL
