Surface-modified PLGA-based nanoparticles that ... - Future Medicine

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Surface-modified PLGA-based nanoparticles that can efficiently associate and deliver virus‑like particles Aim: To design and develop a new nanocarrier appropriately engineered for the adequate accommodation of a virus-like particle, the recombinant hepatitis B surface antigen (22 nm), and intended to be used for the transmucosal delivery of the associated antigen. The nanoparticles consisted of a core blend of poly(d,llactide-co-glycolide) and poloxamer 188, and a hydrophilic shell of chitosan. Results: By by conveniently adapting the nanoprecipitation technique, it was possible to associate a significant amount of active antigen (44%) to the nanocarrier. The resulting nanosystems had a size of around 200 nm and positive zeta potential attributed to the association of chitosan. The nanoparticles were able to deliver the associated antigen in a controlled manner for up to 14 days without compromising its activity, as determined by ELISA. Moreover, the antigenicity of the recombinant hepatitis B surface antigen was preserved for at least 14 days, when stored as an aqueous suspension, and for at least 3 months when converted in a freeze-dried powder. Conclusion: Poly(d,l,lactic-co-glycolic acid)-based nanoparticles represent a promising approach for the delivery of virus-like-particles. KEYWORDS: PLGA-based nanoparticle n rHBsAg n surface modification n vaccine delivery n virus-like particle

The use of biodegradable polymeric nanoparticles for drug delivery has been gaining momentum and has shown significant therapeutic potential [1,2] . Over the last few years, the design of new delivery systems for immunization has received increasing interest and the support of many prominent public and private health organizations, such as the Global Alliance on Vaccines and Immunization (GAVI) and the Bill & Melinda Gates Foundation, among others. In fact, new vaccine strategies able to make vaccination campaigns easier, safer and cheaper have been prioritized and, thus, represent one of the grand challenges in global health. The development of efficient adjuvant and antigen delivery systems is a key issue in order to improve the immunogenicity of vaccines based on purified recombinant proteins. Promising advances for improving vaccine delivery with the application of the polyester-based microparticles, such as poly(lactic acid) (PLA) and poly(d,l-lactic-co-glycolic acid) (PLGA), have been obtained either as needle-free or single-shot approaches [1,3] . A primary obstacle impeding the development of PLGA vaccine delivery systems is the instability of the antigen during the preparation of the delivery system. It has been observed that the antigens not only suffer significant structural alteration (i.e., denaturation, aggregation and degradation) during their encapsulation, but also during the course of their release from PLGA particles upon degradation of the polymer [4–7] . Therefore,

several strategies aimed at preserving the antigen inside the microspheres have been developed [8–10] . Among the different approaches undertaken to overcome this problem, the most efficient have been those based on the formation of core-coated microspheres and PLGA/poloxamer microparticulate blends. In fact, these novel microstructures have been shown to provide long-lasting immune responses against tetanus toxoid [8,9] . It was evidenced that some physicochemical characteristics of these carriers could be further optimized for better performance [11–13] . It was demonstrated that the particle size and the surface composition affect the transport of PLGA delivery systems across the mucosal surfaces. The extent of transporting PLGA-based delivery systems was more important for nanoparticles of 200 nm, although this could also have been influenced by the surface characteristics of the nanoparticles, such as the presence of a hydrophilic corona, such as polyethyleneglycol (PEG) or chitosan (CS), which have improved the interaction of the nanoparticles with the nasal mucosa [11,14] . More recently, as another approach to solving the limitation of the internal acidification within a nanometric structure, nanoparticles made by using an intimate blend of PLGA with polyoxyethylene derivatives, either poloxamines or poloxamers, were developed [15] . This approach was found to be successful for preserving the biological activity of plasmid DNA [16] . Furthermore, intranasal

10.2217/NNM.10.69 © 2010 Future Medicine Ltd

Nanomedicine (2010) 5(6), 843–853

Patrizia Paolicelli1*, Cecilia Prego1*, Alejandro Sanchez1 & Maria J Alonso†1 Department of Pharmacy & Pharmaceutical Technology, School of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain † Author for correspondence: Tel.: +34 981 594 488 ext. 14885 Fax: +34 981 547 148 [email protected] *Authors contributed equally 1

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Paolicelli, Prego, Sanchez & Alonso

administration of blend nanoparticles to mice showed an induction of the immune response whose intensity was dependent on the composition of the blend [17] . Higher and long-lasting IgG levels were induced when blend nanostrutures had a more hydrophilic character [17] . Keeping all this in mind, we hypothesized that an interesting carrier for the delivery of complex antigens, such as recombinant hepatitis B surface antigen (rHBsAg), would be one that preserves the stability of the associated antigen and facilitates the transport across the epithelia. Thus, we selected nanoparticles composed of blends of PLGA and poloxamers coated or not with CS. Some experimental approaches for the efficient association of antigenically active rHBsAg into these nanoparticles and the ability of some selected prototypes to control the release while preserving the antigenicity of the antigen are reported. As a further step, stability studies of the nanoparticles, either as a solution or as a freeze-dried formulation, were performed.

Materials & methods Materials Poly(d,l-lactic-co-glycolic acid) with a lactide/ glycolide ratio of 50:50 (Mw: 14 kDa; Resomer® RG502S) was purchased from BoehringerIngelheim (Ingelheim, Germany). Poloxamer 188 (Pluronic® F68) was obtained from Sigma Aldrich (Madrid, Spain). Ultrapure CS hydrochloride salt (Protasan UP CL 113, with a molecular weight of approximately 125 kDa and an acetylation degree of 14%) was purchased from Novamtrix (Norway). rHBsAg (Mw 24 kDa) was kindly donated by Shanta Biotechnics Ltd (Hyderabad, India). Chitosanase-RD, an N-acetylglucosaminohydrolase prepared from Bacillus spp. PI-7S with an activity of

0.15–0.35 U/mg was purchased from US Biological (MA, USA). ELISA (Murex HBsAg Version 3), a microtiter plate-based sandwich assay using a mixture of monoclonal antibodies specific for different epitopes on the ‘a’ determinant of HBsAg, was obtained from Abbott Diagnostics Division (Abbott, Spain). Antibodies for western blot detection, chicken polyclonal antibody to hepatitis B virus surface antigen and rabbit polyclonal antibody to chicken conjugated with horseradish peroxidase, were purchased from Abcam pcl (UK). All other chemicals and reagents were of analytical grade. Ultrapure water was used throughout the study. Preparation of PLGA-based nanoparticles Poly(d,l-lactic-co-glycolic acid):poloxamer were prepared using the nanoprecipitation technique [15] . Briefly, 20 mg of PLGA and 20 mg of poloxamer were vortexed in 2 ml of methylene chloride for 30 s to obtain a physical blend of both polymers. This solution was poured onto a polar phase (25 ml ethanol) under moderate magnetic stirring, leading to instantaneous polymer precipitation in the form of nanoparticles. The formulation was diluted with 25 ml of water and the stirring maintained for 10 more minutes, in order to collect them as an aqueous suspension. Finally, the organic solvents were evaporated under high pressure. In a further step, PLGA:poloxamer nanoparticles were superficially modified with a hydrophilic coating, the polysaccharide CS, by adapting the methodology previously described. More specifically, PLGA (20 mg) and poloxamer (20 mg) were dissolved in 2 ml of a mixture of ethyl acetate and methylene chloride (ratio 5:1) and immediately

PLGA Poloxamer CS rHBsAg

rHBsAg particles 22 nm

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Figure 1. rHBsAg and loaded PLGA:poloxamer nanoparticles coated with chitosan or uncoated. CS: Chitosan; NP: Nanoparticle; PLGA: Poly(d,l-lactic-co-glycolic acid); rHBsAg: Recombinant hepatitis B surface antigen.

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PLGA-based nanoparticles & delivery of virus‑like particles

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Figure 2. Physicochemical characteristics of PLGA-based nanoparticles. Influence of the recombinant hepatitis B surface antigen loading on the physicochemical characterization of PLGA:poloxamer NPs coated or not with CS. (A) Particle size and (B) surface charge (mean ± standard deviation; n = 3). CS: Chitosan; NP: Nanoparticle; PLGA: Poly(d,l-lactide-co-glycolide).

poured onto 25 ml of a 1:1 (v/v) ethanol/water mixture containing 1 mg of CS under magnetic stirring. The polymers precipitated in the form of nanoparticles and the stirring was maintained for 20 min. Finally, the organic solvents were eliminated by evaporation under vacuum. In order to prepare nanoparticles containing the antigen, two theoretical loadings were selected: 1 and 2% (w/w), with respect to the total amount of PLGA. To achieve the association, the antigen as an aqueous suspension was added to the polymers solution and immediately poured onto the ethanolic phase. future science group

Physicochemical characterization of PLGA-based nanoparticles The hydrodynamic size, polydispersity index (PDI) and surface charge of the nanoparticles were determined by photon correlation spectroscopy and laser-Doppler anemometry, respectively (Zetasizer Nano ZS90, Malvern Instrument, Malvern, UK). All measurements were performed after opportune dilution of the nanosuspension with ultrapure water. The morphology of the nanoparticles was examined by transmission electron micro scopy (TEM; CM 12 Philips, Eindhoven, www.futuremedicine.com

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Figure 3. Nanoparticle transmission electron micrographs. (A) Recombinant hepatitis B surface antigen-loaded poly(d,l-lactide-co-glycolide):poloxamer nanoparticles; (B) a fine detail of the morphology; (C) chitosan-coated poly(d,l-lactide-co-glycolide):poloxamer nanoparticles; and (D) a fine detail of the morphology.

The Netherlands). The samples for the TEM examination were stained with a 2% (w/v) phosphotungstic acid solution. The superficial modification of PLGAbased nanoparticles with CS was determined by elemental analysis. Thus, blank CS-coated PLGA:poloxamer nanoparticles were freezedried, and the solid was analyzed by Elemental Analyzer Fisons (model EA 1108, Thermo Finnigan, Italy). CS content in the nanoparticles was quantified by the amount of nitrogen in the sample. Determination of the association efficiency The amount of rHBsAg associated within polyester-based nanoparticles was directly quantified by ELISA and visualized by western blot, after degradation of the nanoparticles in a medium that did not compromise the activity of the antigen. Poly(d,l-lactic-co-glycolic acid):poloxamer nanoparticles were submitted to a hydrolytic degradation. Briefly, 1 mg of nanoparticles was centrifuged (40 min, 10,000 ×g, 15°C) for the separation of the free antigen. The recollected nanoparticles were hydrolyzed with 0.8 ml of 846


0.75% (w/v) sodium dodecyl sulfate (SDS) in 0.018 N NaOH solution at room temperature for 12 h under shaking, returning the free antigen for the quantive and qualitative analysis by ELISA and western blot, respectively. The degradation of CS-coated PLGA:poloxamer nanoparticles was performed in two steps. First, the nanoparticles were submitted to an enzymatic degradation with chitosanase in order to remove the polysaccharidic coating, and then the polyester core was hydrolyzed. More specifically, after centrifugation of 1 mg of CS-coated PLGA:poloxamer nanoparticles (40 min, 10,000 × g, 15°C), the pellet was suspended in 0.4 ml of 50 mM acetate buffer (pH 5.5) containing chitosanase (0.25 U) and incubated at 37.0 ± 1.0°C for 30 min under moderate shaking. At the end of the enzymatic degradation, 0.4 ml of 2% (w/v) SDS in 0.06 N NaOH solutions was added to the nanoparticles suspension and incubated at room temperature for 6 h under shaking conditions. The free antigen was evaluated by ELISA and western blot. To carry out the ELISA, calibration curves were prepared with blank PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles (without rHBsAg) degraded under the same future science group


conditions. All measurements were performed at least in triplicate and the results reported as mean values ± standard deviation. The immunoreactivity of the linear epitopes of rHBsAg recovered after nanoparticle degradation was evaluated by SDS-polyacrilamide gel electrophoresis (SDS-PAGE) performed under reducing conditions [18] , followed by western blot analysis. For this purpose, free antigen was diluted in Laemmli buffer, heated at 95.0 ± 0.1°C for 5 min, loaded onto a 4% stacking gel (Bio-Rad, USA) and subjected to electrophoresis on a 12.5% separating gel at 200 V until the blue dye reached the bottom of the gel. The gel was then electroblotted to a PVDF membrane in glycine/Tris buffer at 90 V for 90 min. The membrane was blocked with TBST (10 mM Tris, 150 mM NaCl, 0.1% v/v Tween 20; pH 7.5) containing 5% (w/v) skimmed milk powder at room temperature for 1 h, and

then incubated for 12 h at 4°C with 1:1000 diluted polyclonal anti-HBsAg antibody. The membrane was washed five times with TBST and further incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After extensive washing of the membrane, the protein bands were visualized by a chemiluminescence reaction using the ECL Plus Western Blotting Detection Reagents (Amersham Biosciences plc, UK). In the case of the CS-coated PLGA:poloxamer nanoparticles, the degradation process was modified to avoid any interference of the enzyme with the gel. In this case, a reduced amount of chitosanase (0.012 U) was used for CS degradation, whereas the incubation time was prolonged from 30 min to 4 h in order to ensure a complete degradation of the coating. All the others variables of the degradation process were unchanged.


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Figure 4. Antigen associated within PLGA:poloxamer nanoparticles and chitosan-coated PLGA:poloxamer nanoparticles. (A) Association efficiency and loading efficiency of recombinant hepatitis B surface antigen within PLGA-based NPs for two different recombinant hepatitis B surface antigen theroretical loadings (1 and 2%); mean ± standard deviation; n = 3. (B) Western blot analysis of recombinant hepatitis B surface antigen associated within PLGA-based NPs and CS-coated PLGA-based NPs. Lane 1: native recombinant hepatitis B surface antigen; lane 2: rHBsAg associated within PLGA:poloxamer NPs; lane 3: rHBsAg associated into CS-coated PLGA:poloxamer NPs. CS: Chitosan; NP: Nanoparticle; PLGA: Poly(d,l-lactide-co-glycolide).


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In vitro release studies of rHBsAg from PLGA-based nanoparticles In vitro release studies of the antigen from PLGA :poloxamer and CS-coated PLGA:poloxamer nanoparticles were performed in water containing the bacteriostatic agent thimerosal, at a concentration of 5 µg/ml. This mild medium was selected in order to ensure the stability of the colloidal carrier during the study. Thus, nanoparticles were incubated at 37.0 ± 1.0°C under continuous and constant shaking, and predetermined time points (1 h, day 1, 4, 7 and 14), an aliquot was withdrawn and centrifuged (40 min, 10,000 × g, 15°C). Pelleted nanoparticles were resuspended and further degraded to determine the activity of the associated antigen by ELISA, as previously described.

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Figure 5. In vitro release study of rHBsAg from PLGA-based nanoparticles. (A) Release profile of rHBsAg from PLGA:POE and CS-coated PLGA:POE NPs (mean ± standard deviation; n = 3). (B) Western blot analysis of rHBsAg. Lane 1: bulk rHBsAg; lane 2: rHBsAg recovered from PLGA:POE NPs after 4 days; lane 3: rHBsAg recovered from PLGA:PEO NPs after 7 days; lane 4: rHBsAg recovered from CS-coated PLGA:POE NPs after 4 days; lane 5: rHBsAg recovered from CS-coated PLGA:POE NPs after 7 days. CS: Chitosan; NP: Nanoparticle; PLGA: Poly(d,l-lactide-co-glycolide); POE: Poloxamer; rHBsAg: Recombinant hepatitis B surface antigen.

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Freeze-drying of rHBsAg-loaded PLGA-based nanoparticles Aliquots (1 ml) of rHBsAg-loaded PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles suspensions (2 mg/ml) were freeze-dried in the presence of trehalose or sucrose as cryoprotectants at a concentration of 1, 2.5 or 5% (w/v). Therefore, the cryoprotectant:nanoparticle weight ratios were 5:1, 12.5:1 or 25:1, respectively. The suspensions were placed in glass vials and frozen at -80°C overnight. Lyophilization was carried out in a Labconco Freeze Dry system operating at -35°C under high vacuum for approximately 48 h to ensure a dried product. All the samples were reconstituted with 1 ml ultrapure water by simple pipette mixing. Reconstituted nanoparticles were analyzed for their physicochemical properties (size, PDI and zeta potential) and the ability to preserve the antigenicity and structural integrity of the associated antigen were evaluated by ELISA and western blot, respectively. In vitro stability studies of rHBsAg within PLGA-based nanoparticles The stability of rHBsAg within PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles as an aqueous suspension and as a lyophilized product was investigated. Aqueous suspensions of nanoparticles were stored for 14 days and after this time, the nanoparticles were isolated by centrifugation (40 min, 10,000 × g, 15°C), resuspended in ultrapure water and degraded, as previously described. The antigen extracted was submitted to SDS-PAGE followed by western blot analysis. The in vitro antigenicity was also measured by ELISA. Blank nanoparticles maintained in the same conditions as the rHBsAg loaded ones were used as controls for the construction of the calibration curves. Furthermore, the stability of antigenloaded PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles in the freezedried form was investigated. Thus, vials containing rHBsAg-loaded nanoparticles lyophilized in the presence of 2.5% (w/v) trehalose were stored at 4°C for 3 months. Every month one vial was withdrawn and reconstituted with ultrapure water for physicochemical characterization. In additon, the nanoparticles were degraded for further evaluation of the structural integrity of the associated antigen by western blot.

Results & discussion Development & characterization of PLGA:poloxamer nanoparticles The association of rHBsAg within PLGA: poloxamer nanoparticles was achieved using a future science group


simple and mild nanoprecipitation technique. This technique, which does not require the use of high energy sources, was first applied to the nanoencapsulation of lipophilic compounds and, more recently, adapted to the nanoencapsulation of macromolecules, such as soluble antigens into PLA–PEG nanoparticles [11] , and plasmid DNA into PLGA:poloxamer nanoparticles [15] . Based on this information, the nanoparticle preparation was optimized for the nanoencapsulation of a challenging antigen, rHBsAg, which is a macromolecular complex of approximately 22 nm composed of a monomer, lipids and glycidic moieties [19] . By conveniently adapting the preparation conditions, it was possible to prepare PLGA:poloxamer nanoparticles, consisting of a blend matrix of both polymers, and incorporating the 22 nm particulated antigen. Figure 1 illustrates PLGA:poloxamer nanoparticles containing rHBsAg. The particle size increased after associating the antigen in the nanoparticles from 130 to 217 nm, but it remained close to 200 nm irrespective of the antigen loading (Figure 2A) . In all the cases, only one population of nanoparticles was observed (PDI ~ 0.1). With regard to the surface charge, as expected for PLGA:poloxamer nanostructures [11] , negative values of approximately -33 mV were observed. Moreover, the zeta potential values were not affected by the antigen loading (Figure 2B) . In a further step, the surface characteristics of the nanoparticles were modified by attaching the cationic polysaccharide CS (Figure 1) . The formation of CS-coated PLGA:poloxamer nanoparticles was based on the same principle as the uncoated ones. However, in this case, the experimental conditions had to be modified in order to facilitate the entanglement of CS onto PLGA:poloxamer nanoparticles in a single-step process. More specifically, CS was dissolved in the external polar phase, where the nanoparticles formation takes place. As a consequence of the modification of the polarity of the external phase, different organic solvents (ethylacetate, methylene chloride and a mixture of both) had to be tested for their ability to diffuse in the external phase containing CS and, hence, give rise to the formation of nanoparticles. The results showed that the most adequate internal solvent phase for particle formation was a 5:1 (v/v) ethylacetate:dichloromethane mixture. Under these conditions, it was possible to obtain homogeneous nano-sized particles as indicated by laser light scattering measurements (Figure 2A) . Interestingly, the particle size was not modified by the association of the antigen. The effective modification of the surface of the PLGA:poloxamer nanoparticles with CS could be justified by the future science group

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inversion of the zeta potential from negative (-33 mV) to positive (+27 mV) values (Figure 2B) . This zeta potential inversion is a firm indication of the presence of a CS coating on the surface of the particles. However, in order to confirm the CS coating, centesimal analysis of CS-coated PLGA:poloxamer nanoparticles was performed. Results revealed that 17.4% ± 1.2 of CS was incorporated into the nanoparticles. As had occurred with PLGA:poloxamer nanoparticles, the zeta potential was not affected by the association of rHBsAg within the nanoparticles. The morphological examination performed with TEM revealed the sphericity of antigenloaded PLGA-based nanoparticles (Figur e 3) . Uncoated and coated PLGA:poloxamer nanoparticles formed monodispered populations (Figure 3A & C) . The observation showed that both types of nanoparticles are composed of a dense core and a more diffuse superficial layer probably due to the presence of a poloxamer and/or CS corona (Figure 3B & D) . Association efficiency of rHBsAg within PLGA-based nanoparticles A major limitation of PLGA micro- and nanospheres as protein delivery systems is related to the potential protein denaturation during the encapsulation process. In fact, during the formation of polyester-based delivery systems, the drug is exposed to potentially harmful conditions, such as contact with organic solvents and hydrophobic polymer surfaces, which could result in irreversible aggregation or chemical degradation. Therefore, the importance of evaluating 1

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Figure 6. Qualitative analysis of recombinant hepatitis B surface antigen by western blot analysis. Lane 1: bulk recombinant hepatitis B surface antigen; lane 2: recombinant hepatitis B surface antigen recovered from poly(d,l-lactideco-glycolide):poloxamer nanoparticles stored for 14 days at 4°C; lane 3: recombinant hepatitis B surface antigen recovered from freeze-dried poly(d,l-lactide-coglycolide):poloxamer nanoparticles; lane 4: recombinant hepatitis B surface antigen recovered from chitosan-coated poly(d,l-lactideco-glycolide):poloxamer nanoparticles stored for 14 days at 4°C; lane 5: recombinant hepatitis B surface antigen recovered from freeze-dried chitosan-coated poly(d,l-lactide-coglycolide):poloxamer nanoparticles.


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the amount and integrity of the antigenically active rHBsAg associated after degradation of the nanoparticles became a priority. Results showed that the association of active rHBsAg into PLGA-based nanoparticles was not affected by the presence of CS (Figure 4A) . Both PLGA:poloxamer nanoparticles and CS-coated PLGA:poloxamer nanoparticles were able to efficiently associate rHBsAg. However, in both cases, the association efficiency depended on the initial amount of antigen employed, varying between 30 and 44%. In terms of loading capacity, coated and noncoated PLGA:poloxamer nanoparticles duplicated the capacity to accomodate rHBsAg after increasing the loading from 1 to 2%. In addition, in order to assess if the association process altered the structure of the rHBsAg, the antigen recovered after degradation of the nanoparticles was analyzed by SDS-PAGE/western blot (Figure 4B) . The different band intensity observed in the western blot analysis for both samples was due to the different concentrations of the antigen prior to loading the gel. Results revealed a single identical band for the native antigen and the antigen associated within both PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles. Therefore, we could conclude that neither the biomaterials nor the technique for obtaining PLGA-based nanoparticles caused any irreversible aggregation or cleavage of the antigen during the association process.

nanoparticles had no effect on the release profile of the antigen. In both formulations, a burst release after 1 h followed by a gradual antigen release for 14 days was achieved; approximately 90% of antigen was released from the PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles in 14 days. The preservation of the antigen during the release study was attributed to the presence of poloxamer in the formulation. In fact, previous studies have demonstrated the stabilizing effect of poloxamer, which is able to avoid chemical and physical alterations of antigens associated within PLGA-based particles [8,20] , which are frequently described for this kind of delivery system [10,21] . In order to confirm the conservation of the epitope at each time point, western blot analysis of the rHBsAg that remained associated within the nanoparticles at each time point was performed. After 4 and 7 days of incubation, no new bands were detected (Figure 5B) . Thus, the associated antigen did not suffer any structural alterations during the release process, regardless of the nanoparticle composition. At 14 days, almost all the antigen was released, and the visualization of the rHBsAg that remained associated within the nanoparticles was difficult. Overall, from these experiments we concluded that the ability of the PLGA-based nanoparticles developed to preserve the antigenicity of rHBsAg in the course of the release study render them potential candidates for single-shot approach.

PLGA:poloxamer & CS-coated PLGA:poloxamer nanoparticles as controlled release systems for rHBsAg Release profiles of rHBsAg from PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles are reported in Figure 5A . We observed that the presence of a CS coating on the surface of the

In vitro stability studies of rHBsAg within PLGA:poloxamer & CS-coated PLGA:poloxamer nanoparticle suspensions An important issue related to the actual application of new vaccine delivery systems is their capacity for maintaining stability of

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Figure 7. Freeze-drying study. Influence of cryoprotective agents on the size of freeze-dried recombinant hepatitis B surface antigen-loaded PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles; mean ± standard deviation; n = 3. CS: Chitosan; Df/Di: Ratio of mean size of the rehydrated nanoparticles to that of the original nanoparticles; PLGA: Poly(d,l-lactide-co-glycolide).

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the associated antigen during storage. For this reason, it is important to investigate the ability of PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles to preserve the stability of the antigen upon their storage at 4°C. The antigenicity of the rHBsAg associated in unmodified and CS-modified nanoparticles was found to be unaltered for up to 14 days of storage at 4°C (Table 1) . In fact, after this time, it was possible to recover more than 90% of antigenically active antigen from the PLGA:poloxamer nanoparticles and CS-coated PLGA:poloxamer nanoparticles, as evaluated by ELISA after degradation of the nanoparticles. Moreover, western blot analysis corroborated this observation. Furthermore, no structural damage of rHBsAg was observed (F igure 6) .

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Freeze-drying of rHBsAg-loaded PLGA:poloxamer & CS-coated PLGA:poloxamer nanoparticles The chemical and physical stability of PLGA nanoparticles is generally poor in aqueous suspensions during long-term storage [22] , and this is the major obstacle that can limit any further development and application of a colloidal formulation for drug or vaccine delivery. In order to overcome this limitation, both nanoparticle prototypes developed were freeze-dried in the presence of the widely available sucrose and trehalose as the cryoprotectants. Results in Figure 7 show the effect of the different cryoprotectants (trehalose and sucrose) at two concentrations on the hydrodynamic diameter of the nanoparticles after freeze-drying, expressed as ratios of the mean diameter of the rehydrated nanoparticles

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Figure 8. Stability study of freeze-dried PLGA-based nanoparticles with 2.5% of trehalose after storage at 4ºC for 3 months. (A) Physicochemical properties of recombinant hepatitis B surface antigen-loaded PLGA:poloxamer and CS-coated PLGA:poloxamer NPs freeze-dried (mean ± standard deviation; n = 3). (B) Western blot analysis of rHBsAg recovered from freeze-dried PLGA:poloxamer and CS-coated PLGA:poloxamer NPs. Lane 1: rHBsAg; lane 2: rHBsAg recovered from freeze-dried PLGA:poloxamer NPs; lane 3: rHBsAg recovered from freeze-dried CS-coated PLGA:poloxamer NPs. CS: Chitosan; NP: Nanoparticle; PLGA: Poly(d,l-lactide-co-glycolide).



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Table 1. Percentage of antigenically active rHBsAg that remained associated to PLGA:poloxamer and chitosan-coated PLGA:poloxamer nanoparticles after storage at 4°C for 14 days or after freeze-drying in the presence of 2.5 % (w/v) trehalose. Formulation

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91.9 ± 11.0 96.8 ± 2.4 95.3 ± 16.5 92.2 ± 1.3

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Mean ± standard deviation; n = 3. CS: Chitosan; PLGA: Poly( d,l-lactide-co-glycolide); rHBsAg: Recombinant hepatitis B surface antigen.

to the original (Df/Di). Considering that a Df/ Di = 1.0 indicates full recovery of the nanoparticles size, whereas a Df/Di > 1.0 indicates an aggregation, it can be observed, that when increasing the cryoprotectant from 2.5 to 5% (w/v), both formulations could be freeze-dried and reconstituted maintaining their physicochemical properties almost unchanged; furthermore, no significant differences were observed using sucrose or trehalose. In a further step, the reconstituted nanoparticles were also degraded and analyzed for the in vitro antigenicity and structural integrity of the associated rHBsAg by ELISA and SDS-PAGE/western blot, respectively. More than 90% of rHBsAg remained associated with in PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles, thus indicating that the lyophilization did not alter the in vitro antigenicity of rHBsAg (Table 1) . Moreover, the SDS-PAGE/western blot analysis showed that in all cases, the structural integrity of the antigen was preserved; in fact, no additional bands that would indicate the presence of aggregates or fragments were visible (Figure 6) . In a final step, stability studies of the freezedried formulations were performed after storing the powders at 4°C. Every month up to 3 months, the freeze-dried products were reconstituted while preserving the particle size of the nanoparticles (Figure 8A) and, more importantly, the structural integrity of the antigen determined by western blot (Figure 8B) . Therefore, the ability of PLGA:poloxamer and CS-coated PLGA:poloxamer nanoparticles converted into powder formulations to preserve the antigenicity of rHBsAg for at least 3 months could be exploited as a way to improve the thermostability of the vaccine.

Conclusion Poly(lactic acid)-based nanoparticles with suitable properties for the association of particulated antigens were developed. The subunit antigen, 852


rHBsAg, was efficiently associated in nanoparticles of small size with narrow size distribution and superficial charge dependent on the surface composition. These nanoparticles were able to associate a significant amount of rHBsAg in its antigenically active form and deliver it in a controlled manner without compromising its activity. These antigen-loaded nanoparticles could be freeze-dried, resulting in a dry powder that maintained the stability of the associated antigen for at least 3 months. All these features make these biodegradable nanoparticles potential vehicles for improving immunization against the hepatitis B infection.

Future perspective Important efforts have been made in vaccine formulations intended to increase the immunization coverage, after mucosal and/or single-shot antigen administration. A greater, as yet unmet, need, remains in the development of new and safe adjuvants for the delivery of highly purified, soluble antigens as well as virus-like particles. The arguments in favor of the potential of nanosystems are forceful; however, significant formulation improvements are required to overcome the low clinical impact achieved until now using conventional nanoparticles. Within this sense, the engineering of colloidal systems with different core and surface compositions and with fine-controlled characteristics have emerged and become a challenging approach for mucosal vaccination. In fact, it is widely accepted that the core environment of the nanostructures significantly affects the stability of the entrapped antigens, while the surface characteristics affect their ability to overcome mucosal barriers. In addition, nanoencapsulation of antigens can also benefit from a simplification of the vaccination schedules as nanoparticles can offer a control over the release rate, thus enabling the development of single-shot vaccination approaches. New nanoparticulate compositions will certainly alter the landscape of antigen delivery and contribute a step towards the development of more efficient vaccine formulations, thus making vaccination campaigns easier, safer and cheaper. Acknowledgements We would like to thank Shantha Biotechnics Limited (Hyderabad, India) for providing rHBsAg. The advice from Martin Friede of the WHO was greatly appreciated. Cecilia Prego acknowledges a fellow from Angeles Alvariño Programme (Xunta de Galicia). future science group


Financial & competing interests disclosure This work was supported by grants from the Bill & Melinda Gates Foundation. Patrizia Paolicelli would also like to thank the Galenos Network for the Marie Curie Contract MEST-CT-2004– 404992 granted in the framework of the EU Project “Towards a European PhD in Advanced Drug Delivery”. The authors have

Research Article

no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conf lict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary A new nanocarrier specifically engineered for the association and transmucosal delivery of complex protein particles was developed and characterized. The nanocarrier consisted of a core blend of poly (d,l,lactic-co-glycolic acid) (PLGA) and poloxamer and a coating of a mucoadhesive polysaccharide (chitosan). It has a size of 200 nm and a zeta potential that varied dependent on the polymer coating. Using a mild and simple nanoprecipitation technique, it was possible to associate a significant amount of rHBsAg in its active form to the nanocarrier. Neither the biomaterials nor the technique caused any irreversible aggregartion or cleavage of the antigen. Moreover, following adequate dilution in simulated biological media, the nanostructure provided a controlled delivery of the associated antigen in its active form. Finally, the freeze-drying of the colloidal suspension led to the formation of a powder formulation without altering the antigenicity of recombinant hepatitis B surface antigen. This powder form was stable for at least 3 months. Thus, PLGA-based nanoparticles represent promising candidates for improving immunization against hepatitis B infection. Future work will be focused on exploring the potential of PLGA-based nanoparticles as needle-free or single-shot vaccination approaches.

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