ARTÍCULO ORIGINAL

Development of Novel Protocol for Preclinical Monitoring the Release of Adjuvants Encapsulated Mucosal Delivery Carriers

Desarrollo de un novedoso protocolo para el monitoreo preclínico de sistemas de liberación mucosal con capacidad adyuvante

Mohamed Ibrahim-Saeed,^1* Abd Rahaman-Omar,² Mohd Zobir-Hussein³, Isam Mohamed-Elkhidir⁴, Samer Hussein-Al-Ali³, Mothanna Sadiq-Al-Qubaisi², Zamberi Sekawi¹

¹ University of Putra Malaysia, Medical Faculty, Microbiology & Parasitology dept., Serdang, 34300, Malaysia.

² University of Putra Malaysia, Institute of Biosciences, Serdang, 34300, Malaysia.

³ University of Putra Malaysia, Institute of Advanced Technology Serdang, 34300, Malaysia.

⁴ University of Khartoum, Faculty of Medicine, Microbiology & Parasitology dept., Sudan, 11115.

email: zamberi@upm.edu.my

*Virology, Microbiology & Parasitology dept., Medical Faculty, University of Putra Malaysia.

ABSTRACT

This work contributes in vaccines down-stream process by introducing a novel platform for in-vitro monitoring of vaccine-adjuvant delivery profile as a crucial preclinical optimizing step in mucosal vaccines. Nano and micro particles of Calcium phosphate (Cap) vaccine-adjuvant were encapsulated in Chitosan and Alginate polymeric carriers. Adjuvants release profiles monitored in a permeable bag at 37°C, pH 2, incubated in isotonic buffer for 96 hours. The released Calcium in the outer buffer was monitored and compared in-addition to the carrier’s swelling and biophysical properties. The adjuvants and carriers did not interfere with the proliferation of cultured hepatocytes an indicator of their safe use; Chitosan’s viscosity and swelling were higher than Alginate. Chitosan’s Zeta-potential was significantly high positive, while Cap and Alginate were negative. The prepared CaP and Chitosan particles were in nano-size, while the ready-made CaP adjuvant and Alginate were in micro-size using zeta-seizer and scanning electron-micrograph. The release of nano-size particle was in ascending, extended and controlled manner compared to micro-size adjuvant. Moreover, nano-adjuvant release profile from Chitosan was superior compared to Alginate. The core controlling factors in vaccine-adjuvant sustained release includes; smaller adjuvant particles (nano-size), carrier’s low swelling, high viscosity and importantly carrier-adjuvant entrapment reversibility. Chitosan offers sustained ascending superior capacity in releasing Nano-Cap adjuvant. This novel in-vitro pre-clinical study answer a crucial downstream preparative step for optimizing mucosal vaccines before their direct routine in-vivo trial on animal regardless of adjuvant’s particle size or delivery kinetics.

Keywords: nano-adjuvant, delivery carriers, mucosal vaccines.

RESUMEN

Este trabajo contribuye a la investigación de vacunas, a través de una plataforma in vitro que monitorea los perfiles de liberación vacuna-adyuvante, como paso crucial para el desarrollo preclínico de vacunas mucosales. Las nano y macropartículas de fosfato de calcio (Cap), se encapsularon en sistemas de liberación de quitosana y alginato. El perfil de liberación del adyuvante fue monitoreado en membranas permeables a 37ºC, pH2 e incubado en tampón isotónico por 96 horas. Se monitoreó el calcio liberado en el tampón externo y se comparó con la capacidad de hidratación del sistema de liberación utilizado y sus características biofísicas. Los adyuvantes y sistemas de liberación no interfirieron con la proliferación de cultivos de hepatocitos, demostrando un uso seguro. La viscosidad de la quitosana y su nivel de hidratación fueron mayores que los del alginato, mientras que el potencial zeta de la quitosana fue altamente positivo y el del alginato negativo. Las formulaciones de Cap y las partículas de quitosana tenían tallas nanométricas, mientras que el Cap en alginato formó micropartículas que se observaron en zeta seizer y microscopios electrónicos de barrido. El perfil de liberación de las nanopartículas ocurrió de forma ascendente, extendida y controlada en comparación con el de las micropartículas. Además, el perfil de liberación de la quitosana fue superior al del alginato. Los factores esenciales a controlar en sistemas de liberación con capacidad adyuvante incluyen: partículas adyuvantes de pequeño tamaño (nano), sistemas de liberación con bajo perfil de hidratación, alta viscosidad y poder de encapsulación reversible. La quitosana ofrece una capacidad superior para la liberación del adyuvante nano-Cap. Este novedoso estudio, responde a la necesidad de optimizar las formulaciones antes de los estudios in vivo en animales, sin tener en cuenta el tamaño de partículas o la cinética de distribución.

Palabras clave: nano-adyuvante, sistemas de liberación, vacunas mucosales.

INTRODUCTION

Vaccines are preventive biological preparations; they are the only applicable and most effective pre-exposure preventive tools against most of infectious diseases in human. The immune-protective microbial epitope (s), recombinant proteins, or antigen-coding DNA is the core functional ingredient of vaccines. These antigens are the core ingredient responsible for the induction of the different post-vaccination protective immune response; cellular, humoral or mucosal immune responses.

In general, most successful vaccines protect through induction of parenteral immunity mainly (IgG) antibodies. However, protection against mucosal associated pathogens requires induction of more than one type of immune response based on the entry site and the nature infectious agent. Developing effective post-vaccination protection against either nasal or intestinal life-threatening infectious diseases; could only be achieved through inducing both systemic and mucosal immunity. The type of developed post-exposure immune response depends on the site of pathogen entry or the vaccine administration route.

Therefore, the introduction of an improved vaccine delivery system or strategies that maintains the safety issue with a capacity to improve a well-developed post-vaccination protective mucosal immunity became a priority towards mucosal associated pathogens of the digestive, respiratory or the urogenital tracts, through induction of high levels of antibodies, mainly secretory IgA at mucosal surfaces beside systemic neutralizing IgG antibodies. Examples of such pathogens include Streptococcus pneumoniae, Neisseria gonorrhoeae, Vibrio cholerae, Mycobacterium tuberculosis, Human influenza viruses, Human papilloma virus (HPV) (1).

The role of adjuvants in boosting the post-immunization response: Nowadays, vaccine delivery is one of the most strategic approaches used in modulating the post-immunization response of interest. In addition, the mucosal delivery of vaccine became on top of demands, vaccine adjuvants, or delivery carriers are the key tools to be approached for boosting measurable systemic or mucosal immunity, (2). In addition, this could be applied through incorporation of a new improved combination of vaccine with a nano size adjuvants particle encapsulated in delivery carriers. Adjuvants have already been used in vaccines to increase their immunogenicity and to induce higher levels of protective antibody compared with un-adsorbed vaccines.

Generally, adjuvants boost the overall response towards vaccines through potentiating an increased number of lymphocytes clones with minimal antigenic quantity or modulating the type of immune response(s) based on adjuvant capacity to trigger specific lymphocyte and cytokines signaling pathway(s). Adjuvants work through its binding to vaccine epitopes, peptides, or antigens, increasing their molecular weight, delaying their clearance from the circulatory system by the phagocytic cells, improving their antigenic uptake by macrophages, slowing down their clearance by the phagocytic cells, and extending their release to the immune cells. And, in turn controlling adjuvant release could prolong antigen delivery, presentation, activation of a measurable number of lymphocyte clones, and collectively will end-up with an elevated post-vaccination immune-protection. Incorporating nano-size adjuvant in vaccines not only provides an attained availability of vaccine epitope(s) but it will offer a long-lasting protection with increased number of activated lymphocyte memory cells that will expand faster when re-encountering the same pathogen and boost high levels of class specific antibody in term of affinity and avidity compared to the un-adsorbed vaccine (3, 4).

Currently, there are distinctive types of vaccine adjuvants used in human vaccine preparations with countless different properties. However, their chemical nature raises questions about vaccine safety regardless their efficacy. The current trends in vaccine adjuvants focus on developing less toxic, effective, biodegradable, and safer adjuvant or a carrier for vaccine delivery to strengthen the post-vaccination protection against most of human viral and bacterial infections (5).

The advantage of nano-size based vaccine delivery: Since its introduction, large number of Nano-biotechnology, medical applications and protocols had been developed, mainly for studying drug delivery, in which a nano-size carrier particles used to entrap the therapeutic drug and provide a sustainable, slower release of a controlled delivery, in order to achieve the similar therapeutic effect in smaller doses of a prolonged action with minimal drug side effects. Therefore, applying the same principle in vaccine delivery, combining both adjuvant and carriers of a nano-particles size in one formula; could be a useful potential first; to protect the core components of vaccines (antigen, peptides, or epitopes) against degrading effect on vaccine delivery site, such as digestive enzymes, acidity, pH or prolonged exposure to an increased temperature, (6). Secondly; the nano-adjuvants could control the release, boost and modulate the activation of lymphocyte clones to develop the required type and level of post-vaccination immune response.

Calcium phosphate used for long term as a key supplement in bone regeneration, where Cap makes up 70% of bone and 90% of neonate teeth (7). Therefore, it has been considered safe for use in other medical and biomedical applications, such as a supplement for hypokalemia, non-viral gene transfection, biological purification, and delivery of therapeutic protein products like insulin or as an adjuvant for vaccine, (8). Cap is the only nontoxic, biodegradable and non-antigenic adjuvant because it is a body ingredient, compared to other materials used in adjuvant preparation such as aluminium salts. In addition, it has been in use as an adjuvant in vaccines since 1985 (9).

Polymeric nanoparticles used for drug delivery at a lower concentration range between (0.05-1%.), there are so many different polymers used in preparing Nano-particle carriers that improve drug delivery such as gelatines, Chitosan, Alginate, Polyethylene glycol, starch and other carbohydrate derivatives (10). The use of such polymeric carriers could be one of the best choice to improve the delivery of vaccines through mucosal surfaces, where their gradually swelling, increase gel porosity that ends in a controlled release of the entrapped therapeutic protein (11, 12).

Chitosan; a poly-glucose amines, is a biodegradable and nontoxic hydrophobic polymer. Muco-adhesive and of a low solubility in water, therefore it is applied as delivery carrier (13), besides it is also used in preparation of medicinal Nano-composite and as antimicrobial wound healing films (14). Chitosan physical properties, such as viscosity, total high positive charge, particle size, adhesiveness, polymer hydrophobicity and swelling profile play a key role in its improved controlled release of drugs, therapeutic proteins like insulin or its potential application in vaccine delivery mainly towards mucosal surfaces (4, 15). The increased positive charge, which dedicated to its amino group make Chitosan attractive and superior carrier to target the negatively charged mucosal surfaces and very useful in non-viral gene (DNA) delivery (8, 15).

Alginate, a hydrophilic co-polymer, contains α-L-guluronic acid and β-D-mannuronic acid polysaccharide. It is an extract from brown Algae cell wall. It has many useful applications such as a thickening agent in food where it is able to convert a liquid into a gel form at room temperature, it also used in wound dressing, coating of the tablet, tissue engineering, and medical drug delivery (16, 17).

The importance of in-vitro monitoring model in vaccine downstream preparation: In vaccine research generally and specifically the mucosal vaccine development, up-to-date there is a gap in checking the pre-clinical in-vitro delivery (release) profile of vaccine antigens from their adsorbing adjuvants. Therefore, introducing an in vitro delivery model for monitor vaccine release profile from its adsorbing or encapsulating carrier, using in-vitro monitoring protocol is considered a crucial demand and a key tool in designing successful mucosal vaccines. In addition, it will provide an opened in-vitro platform for optimizing vaccine delivery carriers’ formulations to boost and modulate the mucosal immune response based on the interested mucosal site and vaccine administration route.

Vaccine delivery formula: One of the best formulation of choice for improving mucosal vaccine delivery could be achievable through the entrapment of adjuvants in a muco-adhesive polymeric delivery carrier as a potential and novel vaccine delivery formula. Moreover, the in-vitro monitoring of adjuvant or vaccine release from its carriers will serve as a novel and unique pre-clinical protocol for optimizing vaccine delivery profile and formulations of interest and considered as a major additive step in reducing vaccines downstream processing and developmental cost before conducting the routine direct vaccine testing animal trials without un-optimized preclinical in-vitro delivery profile.

Study aim: The aim of this study is to introduce a novel in-vitro delivery protocol for in-vitro monitoring the release and delivery profile of Nano & micro-particles of Calcium phosphate vaccine-adjuvant from their encapsulating Chitosan and Alginate polymeric carriers as a potential new delivery system for mucosal vaccines.

Study design: Calcium phosphate as safe, non-antigenic and biocompatible body ingredient, was chosen as the best material for preparing of Nano-size particles adjuvant. One adjuvant prepared in a Nano-particles size and compared to a ready-made commercial adjuvant of micro-particles size from Brenntag Biosector (Denmark). The two adjuvants used to study their in-vitro capacity in controlling the release profile from a loading carrier of Chitosan and Alginate in a designed in vitro adjuvant delivery model as follows: in-vitro delivery model mimicking oral mucosal permeability, temperature and pH environment were developed using an artificial semi-permeable membrane, the dose response over time used to study both swelling of the delivery carriers and the adjuvants-release profile from their loading two carrier Hydrogels.

MATERIALS & METHODS

Materials: Most of the materials used such as; Calcium phosphate, Chitosan of medium molecular weight (75-85% deacetylated, Cas. Number 9012-76-4), sodium Alginate, (Cas Number 9005-38-3), phosphate buffer tables, dialysis tubes; were bought from Sigma, semipermeable membrane of a mean pore size range between 90-110 nm based on the SEM result from (Spectrum Labs, Taiwan) and Calcium quantification kit OCPC kit from (Reckon Diagnostics Pvt. India).

Preparation and characterization of CAP adjuvant: Preparation: Calcium phosphate (Sigma, USA) 10 mL volume prepared as follows 10 mg/mL (w/v) of the powder dissolved in deionized water; the adjuvant was mixed in a vortex for 3-4 minutes; stirred at room temperature for 90 minutes and sonicated for 45 minutes. Another ready for use commercial CaP adjuvant; bought from Brenntag Biosector, (Denmark), and examined for particle morphology, size, and Zeta-potential.

Preparation of polymeric hydrogels: Chitosan was prepared in gel form as follows; 3% concentration of Chitosan solution (3 mg/mL (w/v)) dissolved in 20 mL volume of 1% of acetic acid in deionized water (v/v), mixed on vortex for 3-4 minutes, centrifuged at 2000 rpm at 4°C for 5 minutes to remove the air. Aliquots of 40 mL were sterilized by autoclave at 121°C for 15 minutes, then the dense gel stirred for three hours and sonicated at high voltage for 15 minutes. The sterile gel preserved at 4°C until used for characterizing tests.

The Alginate prepared in a gel form as follows: 3% concentration of a 20 mL Alginate solution (3 mg/ mL (w/v)) dissolved in 37°C warm deionized water mixed in a vortex for 10 minutes, centrifuged at 4000 rpm at 4°C for 5 minutes. Aliquots of 40 mL tubes, autoclaved at 121°C for 15 minutes, then the dense gel was stirred for 3 hours and sonicated at high voltage for 15 minutes. The sterile gel was stored at 4°C until used for testing for particle characterization.

Physic-chemical properties: Samples of the prepared adjuvant tested for viscosity in digital refract meter (Model AR2008, Kruss, Germany), pH, particle morphology SEM, TEM, particle size, and Zeta-potential measured in Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).The adjuvant and polymeric hydrogels were examined for Carbon Hydrogen Nitrogen Sulphur content; C.H.N.S (LECO, model CHNS-932, USA) instrument. Samples were also examined in Atomic Absorption Spectroscopy; A.A.S for Ca & Na atoms.

Polymers and adjuvants cell cytotoxicity: The cytotoxic effect of the adjuvants was examined in human liver cells after incubation with different concentrations of each of the two adjuvants starting with 1.5 mg/mL; each adjuvant dilution was done in four replicates. The samples were diluted in free serum RPMI-1640 cell culture medium and incubated with Hep-G2 cell line monolayers in 96 well microplate. The plates were incubated at 37°C, in 5% CO₂, for 48 hours with untreated cell as a relative control of (100%) cell viability. The plates were treated with 15 µl of 5 mg/mL Tetrazole solution (Sigma, Cas. Number 298-93-1, USA) including the control cell, the plates were incubated for 3.5 hours at 37°C in in 5% CO₂. The culture media were removed, and the plates were carefully washed with sterile PBS and 150 µL of DMSO were added to dissolve the enzymatic-Tetrazole reaction precipitate, incubated for 15-18 minutes, the plates were agitated and the absorbance read at 590 nm main filter with 620 nm as reference filter. The mean cell viability was calculated in percentage according to the following formula: Cell viability = [Mean O.D of the experimental sample/mean O.D of the control group (after subtracting the reading of blank wells) × 100%].

Adjuvants and carrier gels; pH, Viscosity & Density: pH was measured in a digital hand pH-meter (Sigma (USA) to the Chitosan and Alginate. Samples of Chitosan and Alginate prepared (0.5, 1 and 2.5%) were examined for viscosity in a rheometer (Rotovisco, Germany). The density of CAP, Chitosan & Alginate different preparations (0.5, 1 and 2.5 %) was measured by a refract meter and recorded (r.i). The obtained results were plotted against each sample concentration in Minitab-16 software.

Size and Zeta-potential of the adjuvant and hydrogels: Three aliquots of 3 mL samples of the prepared and commercial Calcium phosphate adjuvants (3 mg/mL), Chitosan (3%), Alginate gels (3%) were used to examine the particle size distribution and zeta potential using Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).

Transmission Electron Microscopy (TEM): Sample volume of five to seven microliters of the preparation and commercial Calcium phosphate, Chitosan and Alginate gels were added to sample holder, and allowed to dry for 25-30 minutes, covered with a negative stain and examined under transmission electron microscope for the particle morphology and size (LEO 912 AB Energy Filtered Transmission. Electron Microscopy (EFTEM) (Carl Zeiss Inc. Germany).

Field Emission Scanning Electron Microscope (FESEM): Small volumes of five microliters from each the two adjuvants were added to the sample holders, after drying for 30 minutes, then samples were gold coated under a vacuum and micrographs were taken under (FESEM; FEI-NOVA NanoSEM 230, Japan) microscope. While the Chitosan and Alginate samples of 1 mL were freeze-dried and the dry sample were added directly to the sample holders, and examined directly without staining for their particulate’s morphology and size under a Scanning Electron Microscope (JEOL JSM6490A, Japan) at a voltage of 20 V.

Preparation of polymeric hydrogels loaded adjuvant: The prepared and commercial Calcium phosphate were entrapped into the Chitosan and Alginate hydrogels as follows: 0.3 mL Nano-Calcium phosphate (10%) added to 2.7 mL of each Chitosan and Alginate gels (3%) at a final concentration of adjuvant 1 mg/ mL of each gel. 1 mL of the commercial Calcium phosphate (3%) was added to 2 mL of each Chitosan and Alginate gels (3%) at a same final adjuvant concentration of 1 mg/mL.

In-vitro carriers swelling & adjuvants monitoring model: The core part of this model is the semi-permeable dialysis tube (spectrum labs, Taiwan), that mimics the permeability of the lining of mucosal surface layers of epithelial cells. It is a weak acid-mucin buffer as follow: (Mucin powder (Cas number 84082-64-4, Sigma-Aldrich) dissolved at concentration of 2%, in phosphate buffered saline (PBS) of a pH 2-3 adjusted with hydrochloric acid under conscious stirring for 60 minute at room temperature. In this development model, 1 mL from each the prepared mixture of adjuvant and carrier were added to a dialysis tube, and each bag was inserted into a well in six-well plate. The well filled with normal saline as isotonic buffer of weak acidic pH 2 was adjusted with HCl, incubated at 37°C water bath under continuous slow agitation at 10 rpm (Fig. 1).

Monitoring hydrogel swelling profile loaded Adjuvant: Nano and micro particles of Calcium adjuvant were loaded into Chitosan and Alginate gel as follows: 3 mL (3%, 2 %, & 1%) of each o