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Effect of Experimental Parameters on
Alginate/Chitosan Microparticles for BCG
Encapsulation

Article in Marine Drugs · May 2016
DOI: 10.3390/md14050090

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marine drugs
Article
Effect of Experimental Parameters on
Alginate/Chitosan Microparticles for
BCG Encapsulation
Liliana A. Caetano 1,2 , António J. Almeida 2 and Lídia M.D. Gonçalves 2, *
1 ESTeSL-Lisbon School of Health Technology, Polytechnic Institute of Lisbon, 1990-096 Lisbon, Portugal;
lacaetano@ff.ulisboa.pt
2 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon,
1649-003 Lisbon, Portugal; aalmeida@ff.ulisboa.pt
* Correspondence: lgoncalves@ff.ulisboa.pt; Tel.: +351-21-7946400

Academic Editor: Paola Laurienzo
Received: 3 February 2016; Accepted: 28 April 2016; Published: 11 May 2016

Abstract: The aim of the present study was to develop novel Mycobacterium bovis bacille
Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface
characteristics and biocompatibility, so that whole live attenuated bacteria could be further used
for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was
encapsulated in chitosan and alginate microparticles through three different polyionic complexation
methods by high speed stirring. For comparison purposes, similar formulations were prepared with
high shear homogenization and sonication. Additional optimization studies were conducted with
polymers of different quality specifications in a wide range of pH values, and with three different
cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge,
physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size
and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative
zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle
suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation
and biocompatibility. Particle formulation consistency was improved when the availability of
functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the
herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by
the intranasal route.

Keywords: alginate; chitosan; BCG; microencapsulation; biocompatibility

1. Introduction
Enhanced immunization strategies must be urgently found for tuberculosis control [1,2]. The current
available vaccine used for pre-exposure vaccination against tuberculosis is Mycobacterium bovis
BCG. As with most vaccines nowadays, BCG is parenterally administrated by subcutaneous route.
This implies a relatively high production cost, the need for cold chain, and the need for trained
personnel for vaccine administration, while it also leads to lower patient compliance. Regarding the
resulting immune response, parenterally delivered vaccines usually produce poor mucosal responses,
which is critical to preventing tuberculosis, as Mycobacterium tuberculosis normally enters the host
through mucosal surfaces. The nasal route might therefore be an attractive alternative administration
route [3].
Regarding tuberculosis, it is essential for a new vaccine to better target the lungs while improving
interaction with antigen presenting cells (APCs) in the lung mucosa, such as alveolar macrophages [4].

Mar. Drugs 2016, 14, 90; doi:10.3390/md14050090 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2016, 14, 90 2 of 30

It is also well known that the eradication of Mycobacterium tuberculosis with pre-exposure vaccination
depends on adequate antigen presentation to amplify the elicited immune response, essentially cellular
Th1 types [5–8]. As such, whole live attenuated bacteria act as the ideal antigen producers and vectors,
as they are multigenic and normally mimic pathogens and surpass natural barriers.
In recent decades, several studies have elucidated the pros and cons of the nasal route for vaccine
administration. It is well known that, for soluble antigens, limited absorption occurs at the nasal
mucosa due to physiological barriers (i.e., mucosal epithelium, rapid mucociliary clearance, protease
degradation) [9]. Many strategies have been proposed in order to surpass these barriers and to increase
the immunogenicity of intranasal delivered antigens, namely, the use of permeation enhancers, mucosal
adjuvants and nano- and microparticulate delivery systems [10,11]. Some studies refer to a boost in
the immune response due to an adjuvant effect of particulate delivery systems, combined with the use
of potent immunopotentiators, either present in the formulation or co-delivered with antigens [12–18].
Taking into consideration the aforementioned, it has been hypothesized that BCG bacilli
modification through encapsulation in polymeric microparticulate delivery systems could be
an alternative to the classical BCG vaccine, suitable for mucosal immunization. Thus, the main goal of
this work was to encapsulate whole live BCG into polymers with biocompatible and mucoadhesive
properties using only mild conditions, so that BCG viability was maintained and the biocompatibility
of the developed microparticulate delivery system was assured. Microencapsulation of BCG in
chitosan-alginate microparticles will allow the following to take place in vivo, in sequence: bacilli
desorption from the particle surface; degradation and erosion of the polymer network; release of
bacteria. Moreover, with the entrapment of BCG in polymeric microparticles, it is expected to change
the BCG recognition pattern by the immune system and to modulate the mechanism of cellular uptake
by APCs cells. The selection of the microsize range was related to the intrinsic length of BCG bacilli rod
of approximately 2–4 micrometers, whereas the preference for electropositively charged microparticles
depends on their ability to better interact with negatively charged mucin [19–21].
The use of biodegradable polymeric particles has been proposed as a promising approach to elicit
adequate immune responses, while protecting antigens from degradation [18]. The preparation of
polymeric particles can be achieved through a wide range of preparation methods, each one yielding
particle formation within a determined size range. For instance, nanoprecipitation and supercritical
fluid technology usually yield nanoparticles, whereas spray-drying and solvent evaporation may
produce nano- or microparticles depending on the experimental conditions [22,23]. It is generally
stated that, for nasal delivery of antigens, nanoparticles are more favorable than particles in the
microsize range, as nanoparticles are better taken up by the M-cells present in the nasal associated
lymphoid tissue (NALT), and better transported through the epithelial cells (by paracellular and
transcellular transference), thus, leading to increased local and systemic immune responses [24,25].
Nevertheless, microparticles sized up to 40 micron have also been described as successful in eliciting
immune responses through nasal administration [11,26–29].
The most commonly described biodegradable polymers are poly(D,L-lactide-co-glycolide) (PLGA)
and poly(L-lactide) (PLA); however, particle formation with PLGA and PLA occurs only in the presence
of organic solvents. This is a major drawback, for several reasons. Not only the use of organic solvents
may lead to relevant toxicological effects, it can also prompt antigen denaturation or hamper cellular
vaccine viability, while formulation methods usually require multiple steps and are time consuming.
In view of the aim of producing a live vaccine, the longer it takes to carry out the formulation steps, the
greater the possibility of losing some of the vaccine or of compromising cell viability, thereby reducing
the encapsulation efficiency and potency of the vaccine.
In this context, chitosan (a deacetylated form of chitin extracted from crustaceans), and sodium
alginate (a natural product extracted from algae belonging to the Phaeophyceae, mainly species
of Laminaria) were chosen to prepare polymeric microparticles by ionic cross-linking methods as
described elsewhere [30–35]. Both chitosan and alginate have been extensively studied as biomaterials
and pharmaceutical excipients due to their biodegradability and low toxicity, and have been included

purity. viscosity. depending on the size. Alginates are block copolymers polysaccharides. Drugs 2016. both chitosan and alginate have been extensively used in the preparation of polymeric nano. the mild preparation conditions of these methods allow the encapsulation of antigens without degradation caused by high temperatures. these components and factors must be matched in order to optimise the overall formulation of alginate microparticles by ionotropic gelation.e. The type of used polymers (i. while being easily adjusted by changing a number of experimental parameters. The widespread use of polyionic complexation methods presents many advantages. usually involving two different polymers and one complexation agent. alginate gel is shrunk and a reduction of the pore size of alginate matrix can be achieved. versatility and flexibility. the polymer to polymer mass ratio. With ionic gelation methods. and other relevant specifications). variables such as the type of polymer or of polymer blends. solubility and temperature.39–44].37]. as the result of the conversion of mannuronic and guluronic acid through neutralization during extraction from its natural source. 90 3 of 30 in the composition of several foods and dietary supplements [36. Gel formation and gel structure are determined by alginate type and calcium salt (Ca2+ ). and production yield. namely. and also that the alginate matrix allows the entrapment of molecules by capillary forces. by adding one polymer solution to the other one with stirring. particles are formed in a single step by a simple mechanism. Furthermore. For instance.and microparticles intended for nasal and oral delivery of vaccines with great results. such as their simplicity. the polymer/polymer and BCG/polymers ratio.e. at lower pH values.34.38]. pH.Mar. Therefore. and to increase both antigen residence time and uptake at the mucosal site. the homogenization type (i. As previously stated. encapsulation efficiency. . As such. thickening and stabilizing properties. Most commonly described complexation agents used with chitosan and alginate are calcium chloride and tripolyphosphate (TPP) [35. being influenced by pH value.25. The herein described effects of experimental conditions on critical features of microparticle formulations provide a processing window for manipulating and optimizing particles in the microsize range for intended applications. which remain free to migrate by diffusion. Alginates constitute a very versatile material.41. as chitosan particles were able to elicit strong systemic and local immune responses to different antigens [16. chemical nature... due to its intrinsic mucoadhesiveness [11. Chitosan and its derivatives are described to increase the absorption of macromolecules through epithelial membranes. particle size. The formulation studies presented in this work aimed the optimization of the preparation conditions of BCG-loaded polymeric particles taking into consideration the final yield of production. speed and duration) and the polymer to antigen ratio are some of the variables which significantly influence the particles’ characteristics. Two other valuable properties of alginates are that they are water-soluble. having numerous pharmaceutical applications due to their gelling. surface charge. Chitosan has been used to prepare nano.45–53]. biocompatibility profile. especially in the case of low G content alginate. The proportion.24.and micro-particles for immunization purposes. It is said that the improved stability of chitosan formulations can be assessed by developing chitosan blends with another polymer. with flexibility increasing in the order GG < MM < MG. shear. particle size distribution and surface charge. the formulation conditions can be changed to obtain desired features. These features make alginates attractive gelling biopolymers for cell encapsulation purposes. While G-blocks provide gel-forming capacity. film-forming. allowing gel formation without heating or cooling. encapsulation efficiency. the type and concentration of complexation agent. as with other commonly used techniques. MM and MG units provide flexibility to the uronic acid chains. molecular weight. oxidation or hydrolysis. polymers solution pH value. distribution and length of these blocks determine the chemical and physical properties of the alginate molecules. as well as the type and time of homogenization procedures and order of polymers and counter ions solutions’ incorporation were studied. composed of long homopolymeric regions of mannuronate (M) and guluronate (G). During optimization studies. namely sodium alginate [36]. 14. being applicable for virtually all polymers which can be polymerized in the presence of a complexation agent.

12:1. critical aspects for vaccine delivery. we confirmed the presence of aggregates mainly in formulations obtained with polymers of high MW (Figure 1). which should be as high as possible. Results and Discussion 2. 14 formulations were initially developed with an alginate to chitosan mass ratio (ALG/CS) ranging from 0. due to chitosan’s and alginate’s mucoadhesiveness. were obtained with chitosan of medium molecular weight when ALG/CS mass ratio ranged from 0. Size Distribution and Surface Charge Previous studies showed that particle size distribution of plain polymeric microparticles prepared by ionic gelation was greatly influenced by the polymers’ mass ratio and molecular weight [20.4:1 to 1:1. viscosity. Characterization of Polymeric Microparticles The purpose of this study was to optimize the experimental parameters to prepare BCG-loaded polymeric microparticles intended for intranasal immunization studies. By visual inspection.5). Regarding the use of low molecular weight chitosan. the conditions for microparticle preparation were optimized during preliminary formulation studies. 2.23:1 (w/w). Using chitosan of medium molecular weight yielded a general increase in particle mean diameter. surface charge. deacetylation degree. Microparticles were characterised for size distribution (mean diameter and span) and surface charge (zeta potential).1. except for formulation F11 prepared with low molecular weight chitosan (span = 9. Best formulations. Moreover. The prepared polymeric microparticles were characterized considering particle size distribution. followed by BCG microencapsulation. with formulation F13 (0. and high viscosity (HV) alginate and high molecular weight (HMW) chitosan. Herein presented formulations were obtained with a relatively narrow size distribution (span ď 5). Drugs 2016. medium viscosity (MV) alginate and medium molecular weight (MMW) chitosan. which in turn might prompt aggregate formation [58.55]. except also for F13 (Table 1). using different combinations of low viscosity (LV) alginate and low molecular weight (LMW) chitosan. Therefore.6:1 to 0. oriented towards obtaining microparticles with a mean diameter of 5–10 µm. thus increasing antigen delivery. G-content.5) (Table 1). Broad particle size distributions can be attributed to the presence of larger single particles. as a model for antigen presenting cells [54. with a narrow and reproducible size distribution. microparticles would be able to promote mucopenetration.57]. and the final yield of production.56. Particle size was the leading assessed property during formulation optimization studies. The effect of polymer molecular weight with increasing alginate to chitosan mass ratio on particle size distribution is presented in Table 1. Therefore. Another key aspect regarding the preparation of vaccine-loaded polymeric particles is encapsulation efficiency.1. . The obtained span values suggest that particles are formed with better consistency when the availability of the functional groups is close to stoichiometric proportion.02:1 to 4. morphology. 14. Biocompatibility of the prepared polymeric microparticles was determined in a cell viability MTT assay. using a human monocyte cell line (THP-1) differentiated into macrophage-like cells. w/w) being the only exception. according to described Methods I and II.59]. Using chitosan of high molecular weight led to an intermediate particle mean diameter. presenting suitable size distribution or surface charge. 2. and with chitosan of low molecular weight when ALG/CS mass ratio ranged from 0. FT-IR studies were conducted in order to assess the interaction between chitosan and alginate ionic groups.1. 90 4 of 30 The expected advantages of the herein described systems for vaccine delivery include the capacity of polymeric microparticulate systems to increase antigen residence time (due to the differentiated release profile in the presence of alginate and chitosan) and to enhance antigen interaction with the cell surfaces. purity) were used to prepare plain polymeric microparticles. particles ranging from 18 to 34 µm were obtained with a narrow size distribution (span < 2.8:1 alginate to chitosan mass ratio. defined as suitable to yield turbid solution without aggregation. Two polymers—chitosan and sodium alginate—with different quality specifications (molecular weight.Mar.

  according  to  the  formula    (d0.9 (diameter the  for which size  distribution  10%.7 ± 1.1 3.4 ± 0. Results are expressed as mean and standard deviation (n≥3).0 n. resulting in  weight led to the formation of smaller particles for the majority of ALG/CS mass ratios.7  n.   Low  39. not determined.4 n.4 ± 1.1 ± 0.  High 51.1  n. The pH of alginate and chitosan solutions was initially  TPP (pH 9.1 ± 0.7 n.d.9  ´26.1  ‐0.6 ˘ 3.  n.6. respectively.1 2.3 +25.3 ˘ 1.2 ± 0. Particle size distribution (mean diameter and span) and surface charge (zeta potential) of 1. and aggregates (▲).2 ± 2.23:1 Medium  Medium 260.9 n. respectively) and span (width of particle size distribution.2  ´10. below.Mar.8:1 led to the formation of microparticles with high positive zeta potential values (+22.1 +47.d.7  mV).0 ± 0.6 ˘ 0. d0.7  1.9 ˘ 1.4 ˘ 0.7  9. more homogeneous particles. n. opalescent/colloidal suspension (■).1 ˘ 1.8 mV).7 ˘ 1.5).6  68. polymers of high molecular weight or viscosity may bind to the surface of such matrices..2 ± 0.  n.7 ± 1.2 +30.3  n.  systems were identified: clear solution (). parameters d0.6:1  Medium  94.1  +16.1 ˘ 0.7  +25.9  4.9 ± 0.5 ± 0.8  ´17.6 ± 0.6 mV to ´26.5 (μm)  d0.6 5.8  3.7 ˘ 4. Higher ALG/CS mass ratios (1:1 and 4.0 ˘ 0. Higher ALG/CS mass ratios (1:1 and 4.1 ˘ 10. Microparticles domain formation using high.  Particle Size.8 n.6 ± 1.5  n.6  36.8  83.5 ˘ 0.d.d.9 ± 1.7 ˘ 1.0  F13  0.6.9 23.7 ± 1.9 and 4.7 ˘ 3.7 ± 3.9 +30.6  2. The  effect  of  polymer  molecular  weight  with  increasing  alginate  to  chitosan  mass  ratio  on  The effect of polymer molecular weight with increasing alginate to chitosan mass ratio on particle particle surface charge is presented in Figure 2.  Low 39.d. Zeta Potential  Zeta Potential Production Yield  Production Yield Formulation  Formulation Span  Span Ratio (w/w)  Ratio (w/w) MW  MW d0. Three different  The pH of alginate and chitosan solutions was initially set to 4.5 ˘ 0.6 ± 0.0 2. Drugs 2016.5 ˘ 41.6  36.7 ˘ 0. F12  0.6 +25.7 +25. F0.0) addition.5 n.7 ± 0.9)/d0.7 ˘ 0.2 ´25. F11-F14.3 4.. resulting in fewer  aggregates. 90  5 of 30  Table Table 1.7 1.9  n. followed by TPP (pH 9.  Figure 1.23:1) led to the the formation of negatively charged microparticles (−10.8 n.2 ˘ 2.7 9.9 and 4.9 ˘ 0.1 ˘ 0. obtained by Method (II) via chitosan pre‐gelation with  addition.  thus.  the size distribution Microparticle  size  parameters d0.9 ± 4.d.d.  F11 0.2 ˘ 0. n.1  +47. Overall. opalescent/colloidal suspension ().6 ± 0.8 ˘ 7.60].d. Formulation F14 prepared with chitosan of low molecular weight was  the exception (+16.6 ˘ 0.7 ˘ 0.5 (µm) (mV)  (mV) (%)  (%)   Low  18.1 n.5  1. the use of chitosan of low molecular weight led to the formation of smaller particles for the majority of ALG/CS mass ratios. Drugs 2016.4:1  Medium  144.3 n.  being  positively  charged.6  83.d.1  3. medium and low molecular weight chitosan.6 mV to  led to the formation of microparticles with high positive zeta potential values (+22.5 ± 0.9 n.   High  51.4 ± 5.3 ± 0.d.7 ± 4.9 mV) with increasing polymer molecular weight. being positively charged.7 ˘ 3. followed by alginate.9  5.d.2 ± 0.7  1.8 3.  respectively)  and  span  (width  of  particle  size  distribution.1 +26.  Low 37.2  +30.9 ˘ 4. matrices.d.1 3.   High  High 68.1  ±  1. obtained by Method (II) via chitosan pre-gelation with alginate.9 ± 0. microparticles on the preparation day.8 ˘ 0.2 ± 0.6  4.5 ± 41.  F12 0.7 ˘ 1.d.6:1 Medium 94.9 (diameter for which 10%.1 ± 1.5 80. The pH of alginate and chitosan solutions was initially set to 4.d. Overall. n. Alginate to chitosan mass ratios ranging from 0.4:1 to 0.d.60].7 ˘ 4. whereas.9)/d0.0 ˘ 0.4:1 Medium 144. Particle size distribution (mean diameter and span) and surface charge (zeta potential) of  microparticles on the preparation day.9 ± 0. Three different systems were identified: clear solution (♦).1 ´ d0.4 4.d. The pH of alginate and chitosan solutions was initially set to 4.6 ‐25. on the the  contrary.1 ± 10.d.1 − d0.4 ˘ 5.d.7 ± 0. forming an outer membrane and leading to increment particle size [33.6.9 mV) with increasing  formation of negatively charged microparticles (´10.d.0  ‐20.4:1 to  surface charge is presented in Figure 2.  n.5  +34.d.8:1 0.4 ˘ 1.d.  Low 18.0 ˘ 8.3 ˘ 0.3 ˘ 0.5 ± 10. whereas.d. The  obtained  results  show  a  greater  influence  of  chitosan  molecular  weight  than  alginate  to  The obtained results show a greater influence of chitosan molecular weight than alginate to chitosan mass ratio on microparticles size distribution.5).  F13 0.1 ± 0. Alginate to chitosan mass ratios ranging from 0.   Low  33.1  n. the use of chitosan of low molecular  chitosan mass ratio on microparticles size distribution. Results are expressed as mean and standard deviation (ně3).6 mV to +47.0 ± 8.0 ´20. This may stem from the ability of chitosan of low molecular weight to diffuse more more promptly in the alginate gel matrix to form smaller.   High  65. respectively.7 139.3 ± 2.9  2. Microparticle set  size4. thus. 50% and 90% of the size distribution falls  according to the formula (d0.8 ± 0.d.  High 65.9 ˘ 0.5 n.6 ± 3. microparticles obtained by Method (I) via alginate ionotropic pre-gelation with CaCl2 followed by chitosan by chitosan addition.4  4.9 ± 1. microparticles.  forming an outer membrane and leading to increment particle size [33. F11‐F14.7 mV).23:1) led to  of high molecular weight) (´0. more homogeneous particles.8  n.8  3.9  and  is characterized using respectively. respectively. and aggregates (N).3 ± 0.6 ˘ 1.8 4.7 ± 4.9 ˘ 0.9 ± 1.8:1  Low Medium  33.  to  4.  ALG:CS Mass  ALG:CS Mass Chitosan  Chitosan Particle Size.3 ˘ 2.5 +34.8:1 Medium 23. 90 5 of 30 Mar.2  3. 14.0  2.d.2 ˘ 0.7 ± 0. 50% and 90% of the size distribution falls below.1 4.5 ˘ 0.1.8 ˘ 0.d.   High  High 107.  polymers  of  high  molecular  weight  or  viscosity  may  bind  to  the  surface  of  such  contrary.9 ‐17.d.1  +26.3  +22.9 and 4. microparticles.1 ˘ 0.9 ˘ 6.1 ´0.   Low  37.6 ‐10. except for one formulation (F13 prepared with chitosan chitosan of high molecular weight) (−0.7 ± 3.1 ˘ 1.1 4.6 ‐26.   Low  25.5  n.0 n. F11  0. medium and low molecular weight chitosan.8 ± 0.8 ± 7.9 ± 6. on  promptly in the alginate gel matrix to form smaller.3 ± 1.3  260.2 3. d0.  except  for  one  formulation  (F13  prepared  with  +47.1  107.5 ˘ 10.2 ± 0.0 ± 0.1.6 mV). not determined.5using  is  characterized  and d0.1 n.  High 80.  F14 1:1   Medium High  139.1  +22.23:1  4.6.6 mV to −26. F0  F0 4.6 ˘ 0.0) addition.2 ˘ 0.6 2.9 ˘ 1.  This  may  stem  from  the  ability  of  chitosan  of  low  molecular  weight  to  diffuse  fewer aggregates.9 ˘ 1.1  +16.    Figure 1.8 mV). 14.  . Microparticles domain formation using high.5 and d0.d.4  +30.5 ± 0.2 ˘ 0.d. microparticles obtained by Method (I) via alginate ionotropic pre‐gelation with CaCl2 followed  F0.8 n.0  F14  1:1  Low Medium  25.

9 the for formulation ±  0. and can in partially due to be decreases. 90 6 of 30 polymer molecular weight. weight  using By using CaCl (“F0_Low”).8:1 0.9 mV). a size  higher it was standard distribution  possible deviation varied  toconsiderably  observe. 0. According to some authors.3–0.9 and 4.6:1 0. been  not size must For the purpose be of greater this study. to chitosan Effect mass ratio of alginate on particle to chitosan masssurface ratio oncharge.4:1 0. chitosan of low molecular weight (“F0_Low”).  and  high  with chitosan prepared with chitosan of low of low molecular molecular weight weight (□). Nevertheless. not shown). with 200 nm to 5 µm being the ideal size [69].7 µm.7  ±  1.7. intracellular partially traffic due  to  ofthe  theincrease  particlesin [62. particle size should be at least 5 µm. in order to enable than 10 µm when phagocytosis is required. with 1:1 ALG/CS By  using  CaCl2  as mass and chitosan of low molecular and chitosan weight (“F14_Low”). this in turn potential as intranasal delivery systems of antigens [64–66]. surface alginate and The pH of alginate and chitosan  solutions  was  initially  set  to  4. mightof BCG bacilli.6:11:1 0. ˘4.  respectively. Drugs 2016.7 molecular  mV).7.7  μm. the authors.5 indicate  0. Effect ofFigureFigure 2. mucosal As uptake size is of particleswhich increased. (∆). Mar. a  reduced  TPP. the  intracellular  microparticles size distribution and traffic  surface of  charge the  particles  profile[62. Carriers the  sample  mass sizing few microns by  weight  have shown higher of  the  microparticles.7. surface charge greatly depends on chitosan  Zeta colloidalin particles in liquid liquid suspension. of the span for and when was chitosan not  microparticles prepared with completely  a given ALG/CS reproducible.5  ±  0. Zeta potential 4. the  (−20.8 ˘to 7. respectively. size must not be greater than 10 µm when phagocytosis is required. it was possible to improve particle size distribution with  a mean diameter of 25. 90  6 of 30  60 60 40 Zeta potential (mV) 40 Zeta potential (mV) 20 20 0 0 -20 -20 -40 -40 0. ALG/CS and ď1.  span  method. This was also confirmed by visual inspection  amino groups.23:1charge  ALG/CS  4.  Zeta  potential  of  microparticles  chitosan solutionschitosan was initially setwas solutions to initially 4. complexation with TPP performed best with 1:1 ALG/CS mass  temperature for days several(datadays (data not shown).to respectively.  and complexation positive  surface  with TPP charge  (+22.3–0.4:1 0.  molecular weight (∆). and chitosan of low molecular weight (“F14_Low”). this inareturn shortmight being  tothe  contribute ideal  size  moderate to rods. and in  These results were important to put into evidence how to modulate the and surface charge profile according to the selected formulation method. Itformation. in order to enable the entrapment of BCG bacilli. which remained suspensions. mass ratio and chitosan of low molecular weight (“F0_Low”). chitosan whereas used to toprepare the alginate chitosanthe mass ratio had an conditions which led to a greater heterogeneity in particle formation. in aincreased givenindicating  thus. 14. chitosan-alginate total  protonated  amino  For chitosan-alginatemicroparticles.  results were thatimportant particle to sizeputdistribution into evidencevaried how toconsiderably and was not size distribution modulate the microparticles completely reproducible. 1  to  40  μm  have  authors. according  F11 to  being the  the selected  formulation  exception).9 ± 0.  Overall.6 mV).8 ± 7. the entrapment of BCG bacilli. The pH of alginate and  alginate 2.  As  size  is  increased. It was also possible to identify the  indicate that the molecular had a major impact in particle size weight of the distribution. 14. For the which can be purpose of this partially due tostudy. Itthe was alginate also to possible chitosan to mass the conditions identify size  distribution  revealed  in  increased  span  values.63].in inalternative presenting  alternative totoTPP. 25. [29]. Zeta  potential  values  provide  a  quantitative  measure  of  the  charge  on  colloidal  particles  in  Zeta potential values providevalues potential a quantitative provide a measurequantitative of the charge measure of on the colloidal charge onparticles liquid suspension. rate as to ranging  According afrom  depot some effect. in alternative to TPP.8 ± 7. Overall.68].  it  was  possible  to  observe. Zeta potential profiles of ±30 mV are described aggregation and stabilize particles in suspension [61]. Results are presented as mean ± SD (n ≥ 3). size must not be greater than 10 μm when phagocytosis is required.7  μm  improve particle (span size particle distribution ≤1.6. particle surface whereas charge.  Carriers  according to sizing  the few  microns  selected have  shown  higher  formulation Particle size is determinant in intranasal delivery and mucosal uptake of particles [29]. Theseindicating method. Particle size is determinant in intranasal delivery and mucosal uptake of particles [29]. Zeta potential profiles of ˘30 mV are described to prevent aggregation and to prevent aggregation andstabilize of  the  stabilize obtained  particles colloidal  suspensions.6 ˘ (+22. to  put  deviation into  evidence  of the how  span to  modulate  the thus.9 prepare  mV).68]. complexation with TPP performed best performed with 1:1 ALG/CS mass of  25.  ALG/CSspan values.  which  remained  stable  without  aggregation  at  room  particles ininsuspension suspension [61].63]. which surface area decreases. with microparticles presenting a reduced with microparticles presenting a reduced mean mean diameter These  diameter of 18. 14. Results are presented presented as as mean mean ˘ SD (n ± SD (n ≥ě3). surface For the purpose of this study. particle size should be at least 5 µm. with microparticles presenting a mean diameter  As for the formulation As method. 90 6 of 30 Mar.7 ≤1. when chitosan of medium and microparticles  high molecular size  distribution  weight and  were surface  used charge (with formulation profile  indicating that particle size distribution varied considerably and was not completely reproducible. Formulation F14 prepared with chitosan of low molecular weight was the exception (+16.6.6 mV).  long slowdown 0. with 200 nm to 5 µm being the ideal size [69]. which remained stablestable without without aggregationaggregation at roomattemperature room for several As for the formulation method. As size is increased. whereas the alginate to chitosan mass  These results indicate that the molecular weight of the chitosan used to prepare the microparticles These results ratio had an important role in modulating particle surface charge.8:14. with microparticles presenting of low molecular weight (“F14_Low”). For chitosan‐alginate microparticles.  mass These  ratio. Mar. 0.2 ˘ 0. groups.  [69]. for microparticles when chitosan of medium and high molecular weight were used (with formulation F11 being the  a broader particle size distribution revealed prepared with exception). particleThe pH ofcharge. molecular  ď1.  medium  molecular  weight  (◌). which are short to moderate long rods. microparticles had a major impact in particle size distribution.  surface potential area  Particle sizeasisintranasal determinant in intranasal delivery systems delivery of antigens and[64–66].4). By 1.7 µm±that 0.9span 0.6  best mV). Overall.  for  ratio had an important roleto in amodulating particle surface charge.6 × 1–4 μm [67.9  and  4.  which  can  be  method.63]. and mediummolecular and high high molecular weight molecular weight (∆).6 ˆ inmuch  the µm 1–4 antigen larger  release particles  [67.68].5 ˘of results  18. particle size should be at least 5 μm. Effect of alginate to chitosan mass ratio on particle surface charge. weight  negative andsurface negative of  the  charge surface chitosan  charge used  (´20.7 charge of  low  ± 1. ititwas mean  was possible diameter  possible of to to improve 18. evidenced as a broader particle  microparticles had a major important impact role in in particle modulating size distribution. a higher standard deviation of the span  led greater heterogeneity in particle evidenced to identify the as a broader particle size conditions which led to distribution a greater heterogeneity in particle formation. with microparticles presenting a mean diameter of complexation agent. with 200 nm to 5 μm  increase in the sample mass by weight of the microparticles.6. This [61]. was also possible whichmicroparticles prepared with a given ALG/CS mass ratio.(˝). and in the potential  as  intranasal  delivery  systems  of  antigens  [64–66]. results  a were  higher important standard of medium and high molecular weight were used (with formulation F11 being the exception). which are short to moderate long rods. it was possible to observe. thus. mass by Carriers weight sizing few microns have of the microparticles.  Zeta surface potential  microparticles. Drugs 2016. this in turn might contribute to a slowdown in the antigen release rate as a depot effect.  size and  negative  distribution with withsurface  4. in order to enable the entrapment  shown area higher decreases. evidenced as revealed in increased span values.  colloidal suspensions. This waswas also also confirmed confirmed by visual by visual inspection inspection of the obtained of the obtainedcolloidal temperature for several days (data not shown). charge greatly profiles  surface depends of  ±30  charge mV  are  greatly on chitosanonto  described  depends prevent total chitosan total protonatedprotonated amino groups.medium molecular weight (◌). According to some  contribute to a slowdown in the antigen release rate as a depot effect.4). a Nevertheless. For suspension.4). massparticle  that  ratio.≤1.23:1 ALG/CS mass ratio and (−20.6 × 1–4 µm [67.2:1 1:1 4. with  2 as CaCl 2 as complexation microparticles  complexation agent.9 mV). Drugs 2016.23:1  µm.2:1 Alginate to chitosanAlginate to chitosan mass ratio (w/w) mass ratio (w/w)   Figure 2. agent.3–0.  the intracellularthetraffic increaseof thein theparticles sample[62. much larger particles ranging from 1 to 40 µm have been . span positive mass  ratio  surface and positive charge surface and  chitosan  (+22. Zeta of microparticles potential of microparticles prepared prepared  with  chitosan  of  low  molecular  weight  (□).9 and set 4.7 the  (span µm (span and≤1. 3).

much larger particles ranging from 1 to 40 µm have been successfully used for intranasal immunization. was chosen to for formulation optimization studies.5 ˘ 0. However. as the size distribution of microparticles with different ALG/CS mass ratios and within the same chitosan molecular weight presented a narrower size distribution.Mar. namely. method consistency and production yield. Therefore. which will help in initial adhesion to nasal mucosa. due to the induced cell integrity loss. Therefore. in order to obtain microparticles of desired and consistent size distribution. the ultimate goal was to encapsulate whole live bacilli of BCG. but also to target the lung mucosa. Drugs 2016. represented by a lower span (Table 2).9 ˘ 0. or. F13 and F14). F12. 14. 90 7 of 30 Nevertheless.9 mV). positively charged particles are expected to be preferable. eliciting good systemic and mucosal responses in mice [9]. particle size distribution and particle surface charge were considered together. Taking into consideration the aforementioned results. different homogenization methods were assessed in four different formulations (F0. Since both high shear and ultrasonication are said to compromise cell viability. The use of chitosan of low and high molecular weights resulted in more consistent and reproducible formulation methods.7) and positive surface charge (+16.8 µm (“F13_Low”) and 14. UT) and ultrasonication (US) used for particle preparation with increasing ALG/CS mass ratios of the final formulation is presented in Table 2. and the 1:1 ALG/CS mass ratio formulation (F14) prepared with chitosan of low molecular weight. In fact. Concomitantly with particle size distribution.6 mV). zeta potential determination allows the estimation of particle suspension stability against subsequent aggregation. The effect of high-speed homogenization (ultra-turrax.71]. and high production yields (>80%) (Table 2). homogenization in an ultrasound water-bath.7 µm (span = 1. one can expect positively charged particles to be preferable to negatively charged ones.7 µm) and narrower particle size distribution (span = 1. two alternative homogenization methods were investigated. alternatively. we selected formulations “F13_Medium MW chitosan” and “F14_Low MW chitosan” for further optimization studies. Surface charge is a critical parameter that affects the mucoadhesion of chitosan/alginate microparticles to the lung mucosa. considering that our proposed microparticulate delivery system must be suitable not only to encapsulate whole live BCG bacteria. it was possible to conclude that the association of 4.23:1 ALG/CS mass ratio (formulation F0) and low molecular weight chitosan provided the formulation’s optimal conditions to obtain polymeric microparticles with smaller mean diameter (+18. although ultrasonication proved to be effective in the preparation of plain chitosan-alginate microparticles. Since mucoadhesive properties of chitosan are mainly explained by the electrostatic interaction and by hydrogen bond of amine groups of this cationic polymer with the negatively charged mucin [36]. simple dispersion with a micropipette. Increasing homogenization times were evaluated. which in turn will prolong the residence time of the vaccine at the site of action. Taking into consideration the obtained results. .8 ˘ 7. also with negative surface charge (´20.4). Effect of Homogenization Method Preliminary formulation studies showed that particle size distribution of plain polymeric microparticles prepared with the ionic gelation methods was greatly influenced by the type and time of homogenization. the overall mean diameter of the obtained microparticles greatly increased (Table 2). The homogenization by ultrasonication led to the formation of microparticles within a narrower and smaller size range.4 µm (“F14_Low”). as ˘30 mV can be an indicator of the particulate systems’ stability [70. with mean diameters between 10. The net positive charge indicates the presence of free surface amino groups in F11–F13 in addition to F14 obtained with chitosan of low molecular weight. When high-speed homogenization was used.2 ˘ 0. with a microparticle size distribution of +25. and regarding particle size distribution.

9 21.1 ˘ 0.8 ˘ 0.7 12.4 µm to “F13_Medium” (0 to 4 min) (Table 3). from 16. Table 3.4 ˘ 0.2 4 19.0 1. The pH of alginate and chitosan solutions was initially set to 4. with increasing homogenization times.1 µm.8 to ´14.0 n.3 ˘ 0.6.1 mV).9 ˘ 0.9 n.1 ˘ 0.2 ˘ 0.5 (µm) (mV) d0.1 n.6.3 3.2 ˘ 0.5 ˘ 3. .6 ˘ 0.0 n.2 mV) and “20 min” in ultrasound water-bath for formulation “F13_Medium” (12.5 ˘ 0.5–21.3 1.d. with formulation “F13_Medium” being the one exception.0 ˘ 0.9 6.0 µm range (Table 3). All microparticles obtained via chitosan precipitation with TPP (pH 9.9 mV). microparticles obtained by Method (II) with modifications.8 ˘ 0. +12.3 ˘ 0.9 ˘ 0.2 ´19.d. Medium and Low refers to chitosan molecular weight and to alginate viscosity.d. 14. and yield of production.6 ˘ 0.6 ˘ 0.6 5.6:1 to 1:1.6 ˘ 1.d.2 ˘ 0.8 ˘ 1.3 n.d. respectively.2 2.5 ˘ 9. microparticles of 1:1 ALG/CS mass ratio that were prepared with low molecular weight chitosan and low viscosity alginate Protanal™.8:1 or 1:1 ALG/CS mass ratios.5 n. thus indicating a good consistency of the used preparation method.9 72.4 ˘ 0.1 2.d.8 ˘ 0. 90 8 of 30 Table 2.1 ˘ 0.2 ´49.0 60.4 ˘ 0.d.1 2. High-Speed Homogenization Ultrasonication Formulation_Chitosan MW Particle Size. microparticles of 0. F12_Low 30.d. Zeta Potential Span Span d0. not determined.5 to 21.9 ˘ 0. MW. The best results were achieved with simple dispersion (“0 min”) for formulation “F14_Low” (12.d.2 1.6 3. The pH of alginate and chitosan solutions was initially set to 4.6 6.d.6 2.2 µm.8 ˘ 0. Drugs 2016.5 F13_Medium. with ALG/CS mass ratios ranging from 0.4 n. Molecular weight. n. 11. Production Particle Size. Particles were overall negatively charged. respectively. Size distribution of microparticles prepared with either 0. .2 4. Size distribution of polymeric microparticles prepared by high-speed homogenization and ultrasonication.0 97.3 to 19.6 ˘ 0. as five formulations exhibited negative zeta potential values (´49.0 n.4 4.8 ˘ 1.2 1.5 ˘ 0.4 ˘ 0.2 +12.0 ´14.8 ˘ 0. microparticles obtained by Method (I) with modifications. 69. presented with a narrow size distribution (low span) (Table 4).9 ˘ 1.5 ˘ 0. F0_Medium 47.4 ˘ 2.5 ˘ 0. Smaller particle sizes were obtained for F14 formulation at 0 and 4 min.5 ˘ 0. F13_Medium F14_Low Time (min) Particle Size. 11. and different chitosan molecular weight.8 ˘ 2. F13_Low 20. 67.5 (µm) Yield (%) F0_Low 34.8 ˘ 0.9 F13_High 52.0 14. Size distribution and zeta potential of microparticles prepared by homogenization in an ultrasound water-bath.9 and 4.5 (µm) (mV) 0 16.7 ˘ 0.8 n.4 ´14.7 F0.0 F13_Medium 65. as such: from 12. F12–F14.d.4 103. F14_Low. Particle mean diameter obtained for both formulations was within the 12.7 ˘ 0.8 6.5 ˘ 1.1 ˘ 10. Particle size increased with increasing homogenization times.2 8.1 2.0 ˘ 0.3 1. 11.0 ˘ 0.0 53.9 and 4. as the stoichiometric proportion of alginate to chitosan of 1:1 might provide a better interaction and nucleation between polymers.0 ˘ 0.8:1 ALG/CS mass ratio prepared with medium molecular weight chitosan and medium viscosity alginate Protanal™. n.8 ˘ 10.7 ˘ 1. probably due to a more favorable ALG/CS mass ratio.d.0 ˘ 0. Production Span Span d0. 10. compared to F13.7 ˘ 2.6 ˘ 0.5 (µm) Yield (%) d0.2 ˘ 0. The use of the ultrasound allowed maintaining particle sizes approximated to the desired particle size (10 µm). leading to smaller sized particles.2 ˘ 0.3 3.0 µm to “F14_Low” (0 to 20 min).1 n.2 15.4 ˘ 0.6 F14_Low 25..6 ˘ 0.Mar. ´14. via alginate ionotropic pre-gelation with CaCl2 followed by chitosan addition.1 ˘ 0. Zeta Potential Particle Size. .7 20 12.7 3. via chitosan pre-gelation with alginate followed by precipitation with 2 mg/mL TPP (pH 9. F0_High 91.2 ´29.6 ˘ 1.0).0) followed by addition of alginate (adapted from Method III).

.0 4.5 11. by simple dispersion with a micropipette for 1 min following additions.0 5. F14.9 12. microparticles prepared with Method (II) presented a size distribution (d0.0 3.7 11. it would be possible to modulate the final surface charge of microparticles.3 ˘ 0.1 43.0 11.3 ˘ 0.0 12.4 3.1 8.0 3.0 ´6.8% 7.2 3.2 1.3 ˘ 1.7 ˘ 0.0 ˘0.1 14.3 11.2 5. 7. The effect of formulation Methods (II) and (III) with decreasing G-content of the sodium alginate polymers used to prepare microparticles is presented in Figure 3.9% Yes 4. low molecular weight chitosan with a deacetylation degree of 92% was used.0 4.0 4.0 5.1 1.0 3.0 5.7 ˘0.4% 4.8 ´23.4 ˘0.6 ˘0. In Method (II). and low viscosity / high-G sodium alginate (ALG) (Protanal™ LF 10/60).0 n.3 3.4 11.0 5. 14.3 ´26.0 15.2 ˘0.4 5.0 18.0 4.7% 6. all approved as pharmaceutical excipient.0 7. Particle Size.8 ˘0.2 ˘ 0. not determined.0 3. the same homogenization method to prepare these microparticles was used.4 6.0 ´3.7 ˘0.6 µm (Manugel™) to 24.0 5.5 ´18.7 µm (Manugel™).5 6.d.2% 5. referred to particles theoretical mass.9 ˘ 5.4634).4 ´25.5% Yes 5.6 15.0 6. and yield of production of microparticles prepared with alginate to chitosan mass ratio of 1:1 (F14_Low).4% 7.0 5.8 ˘0.7 ˘ 0.1 ˘0.4 ´17.3 44.7 1.0 ´2.2 ˘0.4 2.0 ˘ 1.4 ˘0. ranging from 14. ultra-low viscosity sodium alginate of high-G content (63%) Manugel™ LBA.0 4.0 ˘0.8 ˘1.3 31.4 ˘ 6.d.0 4. namely: low viscosity sodium alginate of high-G content (65%–75%) Protanal™ LF 10/60.0 5.0 5.9 ˘ 0.Mar.4 10.4 3.3 ˘0.2 n.0 4.3 ˘0.2 11.3% 3.0 1.3 10.1 6. and it is expressed as mass percentage (w/w).0 5.7% 6.5 13.6 ˘0.3 19.4 1.3% 4.0 ˘0.6 13. Zeta Potential Production ALG pH CS pH F14 pH Span Aggregates d0.0 11.0 4.5 values.1 ´7.5 ˘ 0. Changing the polymers’ addition order by using Method (III) yielded a particle size distribution with a pronounced decrease in d0. three different sets of plain microparticles were prepared using three different commercial brands of low viscosity sodium alginate with distinct G-content.6% 3. *Production yield was determined using gravimetric determination of particles mass following lyophilisation.8 ˘0.0 ´21.3 2. particle surface charge.0 6. Effect of Alginate Type and Polymers Addition Order The results obtained during formulation optimization studies led us towards the rejection of Method (I) and the development of a different preparation method—Method (III). Taking into consideration the aforementioned results obtained for particle size of “F14_Low” prepared by simple dispersion (12.0 ´13.6 ˘ 0. Effect of pH on particle size distribution.0 6.2 ˘0.0 ˘ 16.8 ˘ 0.3 2.0 3.8 ˘0.0 7.0 3.0 6.8 ˘0.5 (µm) (mV) Yield * (%) 3. Results are expressed as mean and standard deviation (n ě 3).3 2. No significant differences were observed between alginates of different G-content within the same formulation method (p = 0.5 ´14.0 11.0 6. The two methods differ in the addition order of the polymers.0 10.1 13.0 16.4 µm (Keltone™).5 ˘0.1 6.3 ˘0.9 ˘0.8 1.5 µm (Protanal™) to 89.9% 7.7 ˘ 0.9 ˘0.4 4.0 4.9 ˘0. 90 9 of 30 Table 4.1 11.4 ´18.6% 6. Regarding particle mean diameter (Figure 3A).9 ˘0.9 16.7 7.4 ˘ 0.5 ˘ 0.8 ˘0.1 ˘0.1 11.2 µm).8 ˘0.0 12.4 ˘0.1 ˘ 0.8 ˘ 0.0 3.0 ´4. As for formulation Methods (II) and (III).0 4.2 14. chitosan and alginate were allowed to interact prior to TPP addition.7% Yes 5.0 ˘0.9 1.4% 5.3 4.4 ˘0.1 n.3 ˘ 0. In order to evaluate the effect of the polymers’ specifications on particle size distribution.0 4.7 ˘ 0.5) from 60.1 ´4.3 ˘0.7 ˘0.9 ˘0.6 13. 3. Drugs 2016. By changing the polymers’ addition order.0 +8. microparticles obtained using formulation Method (III) with low molecular weight/92% deacetylation degree chitosan (CS).5 ˘0.0 17.0 7.2 ˘0.7 ˘0.3 ˘ 0.3 ´22. Chitosan quality specification was kept constant. Microparticles were prepared according to two different formulation methods—Methods (II) and (III)—as described (Materials and Methods section) with modifications.6 31.0 +1. it was possible to identify a bimodal particle size distribution .4 1.6% 6.4% Yes 4. n.1 18.5 ˘ 0.0 ˘ 0.3 ˘0.2 ´16.3 +3.1 ˘ 0.d.1 10.0 4. low viscosity sodium alginate of low-G content (40%) Keltone™ LVCR.d.

prepared with chitosan of low molecular weight and alginates decreasing G-content. pH Value to TPP. in aqueous groups  readily  bind  as a broader acidic solutions.  microparticles  with Method prepared  (II) presented with  Method  (II)  zeta potential values from +14.7 ± 1. Mar. prepared Zeta potentialofof with chitosan lowmicroparticles molecular weight and alginates weight and alginates of decreasing G‐content.3  mV  (Keltone™).6 ˘ 0.  potential reaching  values of ´29. Differences among the two evaluated  statistically significant (p = 0.and Thisthewas also confirmed addition by visual order of the polymers particle size distribution and surface charge than the herein assessed G‐content of sodium alginate.6.6  mV  (Protanal™)  to  −16. For chitosan-alginate These results microparticles.  ALG/CS mass and chitosan of low molecularstudies. (A) Particle size distribution of plain chitosan‐alginate microparticles of 1:1 ALG/CS mass  Figure 3. Particle surface presented  zeta  potential  values  from  +14.(n ± SD 4. the consistent decrease in zeta potential values methods were not statistically significant (p = 0. Results are presented as mean ˘ SD are presented as mean ± SD (n = 3).56. The observed differences were not.9 ± 0. Zeta potential values provide a quantitative measure of the charge on colloidal particles in These  results  were  important  to  evidence  how  the  addition  order  of  the  polymers  plays  an  liquid suspension.6 ± 0.4000). chitosan 3+  groups. microparticles from formulation “F14_Low” (ALG/CS mass ratio of 1:1. prepared with chitosan of low molecular  chitosan solutionsmicroparticles was initiallyofset 1:1to 4.71].  conditions whichcontribute led to a greater since  chitosan  to theheterogeneity solubility protonated  ofinchitosan particle amino evidenced formation. chitosan high pH of alginate.  whereas the anionic nature of alginate (pKa « 3.4–3. These features the bioadhesiveness.7) results from the presence of –COO cationic nature of chitosan (pKa « 6. transport  across    bioadhesiveness.  span values.1  ±  0.0  mg/mL  completely reproducible. statistically significant (p = 0.7) results from the presence of –COO groups.71]. the ´ microparticles had The a cationic major with  impact charge  nature inof density  particle sizeleads depending  chitosan distribution. 14. it charged  is key for surfaces  size distributionchitosan revealed such  in as  mucosal  increased membranes. followed by alginate addition.The  prepared with chitosan of low molecular weight (□). surface charge greatly depends onofchitosan important  role  in were important the  formation  of to evidence chitosan‐alginate how microparticles. statistically significant particle  size  distribution  depending  on  the  used  method. Nevertheless. complexation with TPP performed best with 1:1 stable colloidal suspension. Particle size is determinant in intranasal delivery and mucosal uptake of particles [29].70. and was sets several notof microparticles 1. according to Method (II) (□) and Method (III) (◌). however. Differences among the two evaluated methods were not negative zeta potential values of −29.5) (µm ) Zeta potential (mV) 80 Zeta potential (mV) 0 20 60 -10 40 0 -20 20 -30 -20 -40 0 Protanal TM Manugel TM Keltone TM TM TM TM   -40Protanal Manugel Keltone 0. temperature for Since several days Method (data (III) not shown). optimization weight (“F14_Low”).  mass ratio.9 mV). Regarding Regarding  particle surface particle  charge surface  (Figure charge  3B). it is key for chitosan  with charge density depending on pH value and chitosan deacetylation degree. and in the intracellular traffic of the particles [62. enabled the formation of microparticles with an inferior mean within the  following  As for the formulation a more method. By using CaCl2 as Effectinofalternative complexation agent.0 mg/mL microparticles size distribution and surface charge profile according to the selected formulation method. respectively. Results was initially are presented as meanset to 6. according to Method (II) (˝) and Method (III) ( and pH of alginate. 90 10 of 30 Mar.23:1 ALG/CS mass ratio and chitosan It is well established that anof low molecular weight between ionic complex (“F0_Low”). within a  of sodium alginate. it was possible to improve particle size distribution with It  is  well  established  that  an  ionic  complex  between  alginate  and  chitosan  is  formed  due  to  4.4–3. since chitosan protonated amino groups readily bind to negatively charged epithelial surfaces.7 carboxyl(pKa  µm groups (span ≈  6.6:1 0. and positive surface charge (+22. The (B)  pHpotential Zeta Zeta  of alginate potential  of  plain  and chitosan-alginate of plain chitosan‐alginate microparticles of 1:1 ALG/CS mass ratio. 9.  F11 being theseveral  sets  of  epithelial surfaces.6 mV (Protanal™) to ´16. respectively. Furthermore. the consistent decrease in zeta  suggests thatvalues  potential  a reorganization of the suggests  that  chitosan-alginate a  reorganization  matrix of  the  occurred whenmatrix  chitosan‐alginate  chitosan was allowed occurred  when  to Mar.  in acidic to These  neutral features  solution. Results  molecular weight and TPP solutions (∆). indicating In order tothat assessparticle sizeof distribution the effect varied considerably pH value on microparticle formation. 90 6 of 30 formchitosan was allowed to form a pre‐gel with TPP.7 µm. which can be .1 and 9. according  Figure 2.8:1 1:1 4.  it the polymers seems  plays an that  ALG/CS  total protonatedimportant amino groups. solutions  of  low important molecular  to weight  92% evidence deacetylated  chitosan.9 mV for Keltone™.2:1 Figure 3. role in Zeta the potential formation of profiles of chitosan-alginate ±30 mV are described microparticles.1 ˘ 0.[61].ratio.70.8 ± 7. 60 (B) (A) 100 20 40 10 M ean diam eter d(0. and  for  the  Overall.0.1000).  charge.1≥ and 3). It was also possible to identify contribute to the solubility of chitosan in aqueous acidic solutions.4000). Drugs 2016. it enhancement  was possible of topolar  drugs for observe.particles in suspension homogenization method. membranes. mass ratio.9 andmass ALG/CS 4.0. distribution and which remained surface charge thanstablethewithout aggregation herein assessed at room G-content Since Method (III) enabled the formation of microparticles with an inferior mean diameter.63].6 mV).  The  observed  differences  were  not.  chitosan is formed due to presenting a reduced mean The  cationic  interactions diameter nature the between of 18.7.5)  ≤1. w/w) were preparedthe These results were put into how to modulate using 1. homogenization method. 14.  = 0. microparticles (Figure  prepared 3B).7.The whereas the anionic nature of alginate (pKa ≈ 3.1000). chitosan and TPP solutions was initially set to 6.The of decreasing G-content. Effect ofaccording to  Method  alginate totochitosan Method mass (II)  (solid)  ratio and (II) (solid) and  on particle Method  Methodsurface (III)  (dashed).3  ±  1. reaching Method  (III) negative zeta was  used.5 of  chitosan  ± 0. it seems that ALG/CS aggregation andmassstabilize ratio. withand alginate microparticles interactions between the carboxyl groups of alginate with the amino groups of chitosan [35.Effect of pH Value  span ≤1. ratio had an important role in modulating particle surface charge. As size is increased.  + 3 groups. Nevertheless. thus.4:1 0. 4.  (p however. 90  10 of 30  depending on the used method.  (n = 3). is  conveyed  of alginate and withby  negative thethe  surface positively  amino ofcharge groupscharged  –NH[35. (A) Particle size distribution of plain chitosan-alginate microparticles of 1:1 ALG/CS ratio.56.7. Carriers sizing few microns have shown higher potential as intranasal delivery systems of antigens [64–66].(B) (III) (dashed).9 mV for Keltone™. respectively. more  stable  colloidal  suspension.5) is conveyed by the positively charged –NH− groups.  the addition order So  far. w/w) were prepared using  exception). These results indicate that the molecular weight of the chitosan used to prepare The cationic nature of chitosan leads to the amino group protonation in acidic to neutral solution. it was chosen as the formulation method for the following optimization studies. to  negatively  particle Furthermore.  prepared Alginate with  to chitosan chitosan  mass of  low  ratio (w/w) molecular  weight  and  alginates  of  of decreasing  G‐content. with microparticles presenting a mean diameter of 25.  a pre-gel with TPP. Drugs 2016. followed by alginate addition. 14. anda higher for the standard enhancementdeviation of polarof the spantransport across drugs when chitosan of mediumIn  order  and to  assess  high the  effect  molecular weight of were pH  used value (with on  microparticle  formulation formation. on  pH  value  to the whereas and group amino the protonation alginate chitosan  to chitosan deacetylation  mass degree.3 ˘ 1.4).  inspection have greater impact on of the obtainedparticle colloidal size suspensions.  it  was  chosen  as  the  formulation  method  for diameter.  charge decreased Particle  surface for all formulations charge  when decreased  for  Method (III) was all  formulations  when used.  and  low  viscosity  and  from formulation “F14_Low” (ALG/CS mass ratio of 1:1.  microparticles prepared surfaces with such aasgivenmucosalALG/CS mass ratio.  (−20. Drugs 2016. and the addition order of the polymers have greater impact on So to prevent far. medium molecular weight ◌).3 mV (Keltone™).

4. Samples were prepared in triplicate. both prior to and after freeze-drying.9 mV) (Table 4). The results obtained were analysed by comparing the different pH conditions. At this pH value range.9 ˘ 0.7 span. Drugs 2016.e. and alginate solution pH value within 4.0 resulted in increased particle size (15 to 44 µm) and particle aggregation (Table 4).0 and alginate solution pH = 6. Since a high-G content («70%) alginate (Protanal LF™ 10/60) was used. leading to the formation of stable particles.65) [36]. or trehalose. This might explain why microparticles of lower mean diameter (10. Microparticles were prepared according to Method III with addition of cryoprotectant solution. 90 11 of 30 solutions of low molecular weight 92% deacetylated chitosan.4. Considering the desired particle size distribution (i. Although it is well known that the pH-dependent interaction between alginate and chitosan leads to the formation of stronger complexes at a pH value around 4. The best system was obtained with formulation final pH value of 5. Particle size distribution and zeta potential of samples were assessed for samples without cryoprotectant (batch A) and samples with cryoprotectant (batches B to G).5. w/v) of three different cryoprotectants.0 to 7. with particle mean diameter of 10.3 (Table 4).38) than to guluronic acid (G) residues (pKG « 3. favouring the optimum interaction for the polyionic complex formation. In fact. and narrow span). as mass variations occurred within the sub-milligram or micro-range. The use of both alginate and chitosan solutions with a pH value below 5.2) based on the quantification of chitosan concentration for the determination of the yield of production of microparticles was selected for further studies. .0 (data not shown)..5) have more affinity to alginate mannuronic acid (M) residues (pKM « 3. Effect of Cryoprotectants Addition Table 5 summarizes the different batches of plain “F14_Low” microparticles prepared with two concentrations (5% and 10%.0. as chitosan has a pKa value of « 6. alginate approaches its pKa values. 14. with pH value ranging from 3. It can be observed that particle mean diameter is significantly higher for microparticle suspensions with final pH ď 4. surface charge.0–6. and the amine groups of chitosan are protonated. and a significant part of alginate starts aggregating and precipitating. The obtained suspensions were characterized for particle size distribution.4.4. Aggregation also occurred when chitosan solution pH was beyond 6. the optimal size distributions were obtained when chitosan solution pH value was within 5. leading to the formation of 10–12 µm sized (d0. The production yield was very low (<17%) for all formulations when determined by gravimetry (Table 4).4 µm) and narrower size distribution (span = 2. Within this pH range. and low viscosity and high-G content sodium alginate (Protanal™ LF 10/60). the high degree of protonation of chitosan amino groups prompts a significant reaction with alginate carboxyl groups. therefore suggesting that the Method (III) provides the arrangement of the polymeric matrix in such way that alginate somehow outers the chitosan particulate core. thus. it is also described that the amine groups of chitosan (pKa « 6.Mar. the carboxyl groups of alginate are ionized. and negative surface charge (´18.65. particle mean diameter of approximately 10 µm. which might have contributed to the increased particle mean diameter.0. It would be expected that maximum ionic interaction occurs at a slightly lower pH value for high-M content alginates (such as «60% M-content Keltone™ alginate).5) microparticles. at this pH range. All these formulations presented a negative particle surface charge (Table 4). For that reason. glucose. due to the loss of chitosan solubility. the described method in the Materials and Methods section (Section 3.0–6.9 ˘ 0. Regarding zeta potential results.0.7) were obtained with formulation final pH of 5.4 µm. This is probably related to a low responsiveness of the gravimetric method for the determined mass range.5–5. overall alginate pKa was closer to 3. These particles were prepared with chitosan solution at pH = 5. microparticles prepared with the majority of pH combinations were negatively charged (Table 4). This is probably due to the contribution of alginate carboxyl groups to the negative net surface charge. consisting of sucrose. and yield of production (Table 4).4. a 2.

3 ´19.0 ˘ 0. so that the physicochemical stability of microparticles after freeze-drying can be ensured.3 +13. Regarding particle mean diameter. 4. 90 12 of 30 Table 5.7 +11. Microparticles prepared with 5% and 10% glucose (batches D and E.2 F Trehalose 5% 1:1:120 13. Results are expressed as mean and standard deviation (n ě 3).5 81. or trehalose). indicating a noteworthy increase of particle mean diameter. on production day and following freeze-drying.3 B Sucrose 5% 1:1:120 13. 2. This could also be seen in the exacerbation of the d0.0.2. corresponding to particles with a smaller.2 mV and +14. the obtained low span values (2. with 10% glucose cryoprotectant (batch E) showing better properties after freeze-drying.7 ˘ 4. 54.1 ˘ 9. with average d0. 1:1:0 13.5 +14.3 The pH of alginate. with smaller particle size. the addition of cryoprotectants did prevent some aggregation following freeze-drying.3 ˘ 0.9 ˘ 0. thus. chitosan and TPP solutions was initially set to 6. in average) (Table 5) revealed a high similitude in particle size distribution. It can be concluded that microparticle suspensions were affected by the nature and concentration of cryoprotectants.3 2. Drugs 2016.2 ˘ 1.5 2. not determined. All batches presented similar size distributions. compared to microparticles with no cryoprotectant (batch A). However.0 ˘ 0. glucose.1 ˘ 0.0 3.0 1.6 2.0 ˘ 0.3 2.1 1.2 ˘ 0. The addition of cryoprotectants appears to have contributed to the modification of particle surface charge (Table 5). respectively.5 ˘ 7.9 ˘ 0.9 1.5 ˘ 2. Future studies must be conducted with cryoprotectants in order to optimize particle size distribution and surface charge. particle size distribution.5 values assigned to batches where cryoprotectant had been added (batches B–G). it was within micrometer range for all prepared batches (Table 5). respectively) presented a positive surface charge. 10% glucose and 5% trehalose.1 n.2 D Glucose 5% 1:1:120 12.4 ˘ 0.6 1.6 ˘ 0. Characterization of plain chitosan-alginate microparticles (formulation “F14_Low”) batches without (batch A) and with (batches B to H) cryoprotectants addition (sucrose.Mar.5 2. particle size distribution profile changed and all batches presented up to 10-fold increased d0.3 ˘ 1.5 Span A . as in the case of “F14_Low”.d.6 ˘ 1.5 Span (mV) d0. after freeze-drying.3 ˘ 0.3 93. as microparticles with no cryoprotectant (batch A) presented negative zeta potential values (´19.9 ˘ 0.1 G Trehalose 10% 1:1:240 12.5 ˘ 0. 65. 14. with zeta potential values between +11. probably due to the formation of larger particles or particle aggregates.0 n. and narrow.6 ˘ 50.3 E Glucose 10% 1:1:240 13.1 C Sucrose 10% 1:1:240 13.5 µm.6 ˘ 1.d.5 values of 13.1 n.1 ˘ 2.0 2.8 2.d.1 ˘ 0.2 1. As for particle size distribution width. suggesting that microparticle preparation was reproducible. suggesting that addition of cryoprotectants did not influence particle size for batches prepared under thesame conditions.. Nevertheless.5 values (Table 5).4 1.5 ˘ 0.7. Polymer–Polymer Interaction by FT-IR Analysis Formation of microparticles of chitosan with alginate is a result of strong interactions by hydrogen bonds between the functional groups of the polymers in which amino and amide groups present in . for 5% sucrose. n. It has been described that slightly acidic sucrose and glucose generate a good isotonic medium (in terms of electrostatic stability) for negatively charged particles. thus.4 ˘ 0.3 mV.d. E and F.6 ˘ 0. these additives reverse zeta potential [72].9 ˘ 15.7 mV). low span and average zeta potential positive value.7 170. This is probably due to the adsorption of the molecules to the surface.9 populations for all samples after freeze-drying (data not shown).0 ˘ 0. but for positively charged particles. as batch A (particles with no cryoprotectant) presented the highest particle mean diameter.8 ˘ 0.8 ˘ 0. with approximately two-fold higher d0. it was possible to observe that there were no significant differences concerning particle size distribution (Table 5).2 47.5 values and low span values. with lower d0. thus. whereas microparticles prepared with cryoprotectants (batches B. With samples analyzed before freeze-drying (on production day).1 and 9. Before Freeze-Drying After Freeze-Drying Cryoprotectant ALG:CS:Cryoprotectant Batches Particle Size (µm) Zeta Potential Particle Size (µm) % (w/v) Mass Ratio (w/w) d0.4 ˘ 0.1.0 ˘ 0.8 ˘ 0.1 ˘ 8.9 ˘ 0. 63.5 µm.5 ˘ 15. respectively) performed best.

 FT‐IR spectra of plain chitosan‐alginate “F14_Low” microparticles (1:1 ALG/CS mass ratio)  Figure 4. and  macromolecules  [53].   Figure 4. 90  13 of 30  bands of the amino groups.  The  cm observed  thechanges  amino peak from 1559 tobands  1533 cm ´ 1 ´ 1 in  the  absorption  of  the oramino  1560 cm at carboxyl  groups.  distribution observed in microscopic images was consistent with that obtained by laser diffraction.  partial N-deacetylation of chitin). The  FT‐IR  spectrum  of  microparticles  produced  with  final  pH  of  4. and amide bonds [35].  Microparticle morphology was characterized by microscopy. the ‘simple dispersion’ method was chosen for further BCG encapsulation. particle sizediffraction.0 cm 1414  and −1. 90 13 of 30 Mar. with cm 1559  to  1533  the−1 amide or  1560 peak cm−1 from at  pH  1641 4.0) and 1 mg/mL Protanal™ sodium alginate that a suitable formulation of BCG‐loaded microparticles can be developed and further assessed in  (pH = 6.  a few steps. Surface Morphology  2. suggesting an effective interaction between polymers at pH 4. Additionally. As a result. there are changes in the FT-IR spectra in the absorption bands of the absorption  amino groups. respectively. Based on the identification of  chitosan take part. groups.  under  mild  studies. complexation. 1613 1613  (symmetric  (symmetric COO´COO −  stretching  vibration). Bands wave numbers (cm with increasing pH of the microparticles suspension.  FT‐IR  analysis  was  able  to  illustrate  changes  in  the  wave  number  FT-IR absorbance  analysis in  to was able the  region  changes illustrate of  amino  and wave in the amide  group and number vibrations  with  absorbance in increasing  the region ofpH  of  the  amino and microparticle suspension (Figure 4). revealingConsidering  homogeneous the populations results  obtained  during  of narrow formulation  particle optimization  size distribution studies. Drugs 2016.0  into and  singlet band at 1609 5. after complexation  with  chitosan. and 1415 (asymmetric COO − stretching vibration).  chitosan.  The observed and  amide  bonds  can  be  changes in the absorption bands of the amino groups. 2.  5.  bond).  pH 4. under mild conditions and only requiring vaccine production.4). Surface Morphology Microparticle morphology was characterized by microscopy.3. 14.0) and 1 mg/mL Protanal™ sodium  Considering the results obtained during formulation optimization studies.  This  formulation  was  chosen  because  it  allowed  the  formation  of  formulation of BCG-loaded microparticles can be developed and further assessed in immunization microparticles  of  suitable  mean  diameter  and  surface  charge  without  aggregation. and amide bonds canof be attributed  to  an  ionic  interaction  between  the  carbonyl  group  of  alginate  and  the  amino  group  chitosan. critical for future sterile production during vaccine production.1.7 reveals alginate respectively.0 and 5.Mar. respectively.0  and  5. thus.  amide group vibrations with increasing pH of the microparticle suspension (Figure 4).  Both  chitosan  peaks  were  similarly  shifted  by  a  few  cm   after  −1 complexation with chitosan. 14. Both chitosan peaks were similarly shifted by a few cm ´1 after complexation with alginate. Both F13 and F14 microparticles particle size distribution observed in microscopic images was consistent with that obtained by laser  presented regular and smooth surfaces related to a generic spherical shape (Figure 5). bands  carboxyl concerned  with amide groups. and 1415 (asymmetric COO´ stretching vibration).4). carboxyl groups.7. Both F13 and F14 microparticles  presented regular and smooth surfaces related to a generic spherical shape (Figure 5). Drugs 2016.7. produced with 1 mg/mL low MW chitosan (pH = 5. and the  vibrations  bonds [35].  the  ‘simple  dispersion’  method  was  chosen  for  further  BCG  encapsulation. and the  −1 complexation ´1 .0 and 5.7  reveals  alginate  The FT-IR carboxyl  spectrum peaks  slightly of microparticles shift  from  1613  and  produced 1415  cmwith −1  to  final 1609  pH and of 4. thus. suggesting an effective interaction between polymers at pH 4. .3. formulation.7. so that a suitable immunization  studies.7. FT-IR spectra of plain chitosan-alginate “F14_Low” microparticles (1:1 ALG/CS mass ratio) with increasing pH of the microparticles suspension.  1569  (strong  protonated  amino  stretching vibration).  F14_Low  (Figure 6).  so  produced with 1 mg/mL low MW chitosan (pH = 5. This formulation was chosen because it allowed the formation of microparticles of suitable conditions  and  only  requiring  a  few  steps. with the amide peak from 1641 into singlet band at 1609 cm .of Based functional  on thegroups  present ofin  identification CS  and  ALG  absorption bands concerned with the vibrations of functional groups present in CS and ALG macromolecules [53].1. alginate  (pH =  6. F14_Low formulation. revealing homogeneous populations of narrow particle size distribution (Figure 6). and amino  peak withfrom alginate. Additionally. 1569 (strong protonated amino peak—from peak—from partial N‐deacetylation of chitin).  attributed toThe  peak  absorbance  an ionic interaction of  amino the between groups  of  chitosan  carbonyl group at  of1153  cm−1 and alginate was thealso  present  amino after of group The peak absorbance of amino groups of chitosan at 1153 cm´1 was also present after complexation. Bands wave numbers (cm´1) are as follows: 1641  −1 ) are as follows: 1641 (amide  (amide bond).  respectively.0 and 5.  critical  for  future  sterile  production  during    mean diameter and surface charge without aggregation. carboxyl groups.  after  carboxyl peaks slightly shift from 1613 and 1415 cm ´1 to 1609 and 1414 cm´1 .

90 14 of 30 Mar. low according to Method (III) by chitosan pre‐gelation with TPP followed by alginate coating. low  according to Method (III) by chitosan pre-gelation with TPP followed by alginate coating. chitosan. (B) Contrast phase micrograph (40×) of “F14_Low” microparticles (1:1 ALG/CS) prepared according to Method (II) with  micrograph (40×) of “F14_Low” microparticles (1:1 ALG/CS) prepared according to Method (II) with  micrograph (40ˆ) of “F14_Low” microparticles (1:1 ALG/CS) prepared according to Method (II) with chitosan of low molecular weight. A different macroscopic behavior of cell  led to partial and ephemeral aggregation.BCG  BCGstrains  strains were  were suspended different media.  and and (F13). Particle size distribution of microparticles produced with alginate to chitosan ratio of Figure 6. weight chitosan.  Pasteur  rBCG-GFP the  and  bacilli presented surface  charge  rBCG‐GFP  BCG  predominantly of presented  bacilli  Pasteur  negative appears  to  be  predominantly  zeta slightly  only  negative  potential values more  zeta  potential  (Table values  6). LMW. 5. 90  14 of 30  (A)  (A)  (B)(B) Figure  Figure 5.  bacilli  presented  predominantly  negative  zeta  potential  Both values  BCG Both (Table  Pasteur BCG 6). In order effect  of  experimental  conditions  on  BCG  bacilli  surface  charge. which led to partial and suspension was also distinguished—BCG Pasteur suspension formed a fluffy surface layer.9% NaCl was heat killed as it is described in in the Materials and Methods section (Table 6).  charge of BCG surface  Pasteur charge  appears of  BCG  to be appears  Pasteur  only slightly more electro‐negative than rBCG‐GFP for all tested conditions.  HMW.Mar.  chitosan. 14.8:1 0. Particle size distribution of microparticles produced with alginate to chitosan ratio of 4. In order to assess the  to assess in  the effect of experimental of  experimental  conditions conditions  on on BCG BCG  bacillisurface  bacilli  surfacecharge. Particle size distribution of microparticles produced with alginate to chitosan ratio of 4. 14. the surfacethe  Overall. high  high molecular  molecular molecular  weight  weight chitosan. LMW.  2. this phenomena was not observed for rBCG‐GFP strain.9% NaCl was heat killed as it is described  effect  suspended  in in  different media.  MMW. whereas BCG previously suspended in 0.2. 6).  F0  F0 (F14). In order to assess the  bovis BCG Mycobacterium bovis  BCG  Pasteur and rBCG-GFP strains was measured at low electrolyte concentration. (F0).  HMW.8:1 (F13).  mediummedium  molecular  molecular molecularweight  weightchitosan.  F13‐F14  microparticles  prepared  according to Method (III) by chitosan pre‐gelation with TPP followed by alginate coating.8:1  (0. zeta zeta potential of Mycobacterium potential of to Pasteur and rBCG‐GFP strains was measured at low electrolyte concentration.  charge. low  molecular weight weight chitosan.  with  CaCl F0   microparticles  followed  by  prepared  chitosan  according  coating. F13-F14 microparticles prepared 2 ionotropic  pre‐gelation  with  CaCl2  followed  by  chitosan  coating. Encapsulation Efficiency  2. Drugs 2016. 90  14 of 30  Mar. 14.8:1  ALG/CS)  ALG/CS) ALG/CS)  prepared according to Method (III) with chitosan of medium molecular weight. Encapsulation Efficiency 2.(Table  Overall.     Figure 6. 4.  high  molecular  weight chitosan.9% NaCl was heat killed as it is described  the Materials Both  BCG  and Pasteur  Methodsand section (Table 6). A different macroscopic behavior of cell  to  be  electro-negative only  slightly  more  suspension was also distinguished—BCG Pasteur suspension formed a fluffy surface layer. MMW. Therefore. this phenomena was not observed for rBCG‐GFP strain. led to partial and ephemeral aggregation. Therefore. which    ephemeral aggregation. Figure  (A)  (A) Polarized  Polarized light  Polarized light micrograph  light micrograph (100×) of  micrograph (100ˆ) (100×)  of “F13_Medium”  of “F13_Medium”  “F13_Medium” microparticles  microparticles microparticles  (0.23:1  Figure 6. this phenomena  was not observed for rBCG-GFP strain. charge.8:1 (0. A different macroscopic behavior of cell suspension was electro‐negative than rBCG‐GFP for all tested conditions.  Therefore. (A) 5.  also distinguished—BCG Pasteur suspension formed a fluffy surface layer.  chitosan.  MMW. microparticles  prepared  microparticles preparedaccording  accordingto  Method  (I)  by  to Method alginate  (I) by alginate (F0). Drugs 2016.23:1  (F0).23:1 0.HMW. rBCG‐GFP  in the Materials and Methods section (Table 6).  chitosan of low molecular weight.  different media.  F13‐F14  to  Method  (I) prepared  microparticles  by  alginate  ionotropic pre-gelation with CaCl2 followed by chitosan coating. which  than rBCG-GFP for all tested conditions. Drugs 2016. (B) Contrast phase  prepared according to Method (III) with chitosan of medium molecular weight.8:1  (F13). Encapsulation Efficiency  The ability of chitosan/alginate microparticles to encapsulate Mycobacterium bovis BCG depends  The ability of chitosan/alginate microparticles to encapsulate Mycobacterium bovis BCG depends The ability of chitosan/alginate microparticles to encapsulate Mycobacterium bovis BCG depends  to  a  great  extent  on  bacteria  surface  charge.      .  medium  molecular  weight  chitosan.  chitosan of low molecular weight. LMW.  BCG  strains  were  suspended  Pasteur and rBCG‐GFP strains was measured at low electrolyte concentration.  zeta  potential  of  Mycobacterium bovis  BCG  to aa great great extent extent onon bacteria bacteria surface surface charge. (B) Contrast phase  prepared according to Method (III) with chitosan of medium molecular weight.  ionotropic  and  1:1  pre‐gelation  (F14). whereas BCG previously suspended in 0.  and Overall.  0.2. 1:1  1:1 (F14). whereas BCG previously suspended in 0.2.

Drugs 2016.7ˆ107 CFU/mL for microparticles prepared by Method (III) (Table 7). Preliminary formulation studies revealed that particle size distribution and surface charge were influenced by the polymer to polymer mass ratio and the formulation method.9 ˘ 2. Some hydrophobic interaction involving lipid within the surface may also be involved.2 ˘ 2.9% NaCl. referred to as plain microparticles (Table 7). an inversion of zeta potential values occurred.4 ˘ 11. and with BCG loads of 8. with modifications..5 ´23. complex structure that surrounds the bacillus and is thought to play a critical role in the pathogenicity of Mycobacterium tuberculosis.9 ˘ 11. with increasing BCG loads of 8. water. bovis BCG would be obtained by suspending BCG bacteria in chitosan at a pH below its pKa (e. Other mechanisms.1% low MW chitosan +85. at least partially. when BCG bacilli from either strains were suspended in low molecular weight chitosan. Zeta Potential (mV) Inactivation Method Medium BCG Pasteur rBCG-GFP H2 O ´39. Surface charge of inactivated Mycobacterium bovis BCG (strains Pasteur and rBCG-GFP) bacilli suspended in different media. As such. BCG encapsulation led to decreased particle size in comparison with plain microparticles.025% low MW chitosan +83.6 ˘ 1. as well as for sensing processes. since the mycobacterial cell envelope is a lipid-rich. The negative surface charge for cells of all Mycobacterium BCG species arises from the phosphate groups of phosphodiester linkages between the peptidoglycan and the arabinogalactan of the basic cell wall structure which is common to all species of Mycobacteria [73]. in a concentration dependent fashion.3.5 10 mM PBS ´20. 14. In opposition.g.0 0. A large number of mycobacterial lipoproteins have been suggested to be important components for the synthesis of the mycobacterial cell envelope.7 ´21.7 ˘ 12. 151 ) 0.7ˆ107 . Table 7 summarizes the different batches of BCG-loaded “F14_Low” microparticles that were prepared and the obtained results.1 +92.0 ´32. such as hydrophobic interactions. In this way. pH = 5). might also be involved in bacteria microencapsulation. particle size increased in a concentration dependent fashion.1 ˘ 11. protection from stressful factors and host–pathogen interactions [74. Whether BCG microencapsulation would have a great impact on microparticles features was uncertain.3 ˘ 1. Additionally.7 ˘ 1.6 ˘ 3.9 ˘ 3. .9 The nature of the adsorbing species on the cell surface of the two strains might explain the obtained variations. 1. mediated by ionic interaction between bacilli and chitosan. However. thus these parameters were investigated.4 ˘ 1.3ˆ107 CFU/mL for microparticles prepared by Method (II).1).3ˆ106 and 1. BCG-loaded “F14_Low” microparticles (1:1 ALG/CS mass ratio) were prepared as described in the Materials and Methods section (Section 3. For microparticles prepared according to Method (II). and 3. a good encapsulation efficiency was sought. Results are presented as mean ˘ SD (n = 3). it was hypothesised that the greatest encapsulation/association efficiency for M.4 Temperature (80 ˝ C. The encapsulation efficiency was also determined.5 0. so that the polymer is predominantly positively charged.5 +90. Within BCG-loaded microparticles.9% NaCl ´29.3ˆ106 .5 ´13.75]. BCG encapsulation led to increased particle size.Mar. for microparticles prepared according to Method (III). Taking into consideration the abovementioned. we chose to entrap monodisperse bacteria in chitosan microparticles by means of controlled gelation of chitosan with TPP followed by alginate addition.0 Cell culture medium ´27. or cell culture medium. Zeta potential profiles showed no major differences between bacilli suspension in either 0. 10 mM PBS. suggesting that the mechanism of association of the bacteria to chitosan is. 90 15 of 30 Table 6. by Method (II) or Method (III).

4 F III .6 mV.7 4.0 84.9 ˘ 5. thus. Figure 7B confirms that viable cell density decreased approximately 1 log after 3 weeks for BCG-loaded chitosan-alginate microparticles and for BCG suspended in 0.2 ˘ 2.8 ´13.8 ˘ 0.0 ˘ 4.7 ˘ 2. it is extremely difficult and challenging to achieve efficient.9 87. Due to the extremely high content of complex lipids present in the BCG cell wall. Regarding BCG Pasteur.9 – G III 1. For rBCG-GFP. indicating that encapsulation occurred.1 ˘ 0. Drugs 2016.0 ˘ 3.4 ˘ 2.3 ˘ 4.4 ´12.1 – H III 8. was not determined.) and occred in a concentration dependent fashion.5 70. The encapsulation mechanism. the same effect was observed in the control groups of 0. Method (II) produced microparticles (both plain and BCG-loaded) that were electropositively charged.025% chitosan.6 ´16.E.0 6.3ˆ107 36.2 13.4 ˘ 2. The encapsulation of BCG Pasteur into microparticles was efficient (70%–87% E.9 mV to ´16.0 ˘ 3.4 J III 3. These results indicate that negatively charged BCG bacilli is present.9 ˘ 2. BCG cell viability following suspension in chitosan was investigated over time by a colony-forming units (CFUs) assay.5 ˘ 2.8 ˘ 2.6 – B II 1. (%) Method Load (CFU/mL) d(0. Overall.7ˆ106 35.5 ˘ 12. Both strains BCG Pasteur and rBCG-GFP were assessed in this cell viability study.0 ˘ 1.8 ˘ 1. BCG Cell Viability Chitosan is described as having antimicrobial potential [48–50].1 mV. 18. two different patterns were obtained. Zeta Potential Batches Span E. although the suspension of BCG in 0. These effects were observed for both strains regardless of the suspension media (p > 0.7 ´3.0 ˘ 3.8 ˘ 2.5 14.0 ˘ 2. and approximately 3 log on the 6th week. .6 C II 8.5 85.9% NaCl-suspended BCG. 90 16 of 30 Table 7.3 74.3.3 ´12.6314) in cell viability losses between BCG-loaded microparticles and BCG suspended in 0. 2. as there were no statistically significant differences (P = 0.7ˆ107 22.7 Regarding particle surface charge. 60.8 E II 3.5 ˘ 1. 14.8 12.3 2.Mar. uniform and reproducible microencapsulation experiments.7 ˘ 2. Overall.6 83.2 3.1 11.0 ˘ 6. chitosan may not be considered cytotoxic at the tested concentration.2 ˘ 0.4 2.5 ˘ 2. with zeta potential values ranging from ´3.3.3ˆ106 39.1). and approximately 2.7ˆ107 41. Whether BCG suspension in chitosan would compromise BCG cell viability was uncertain.0 25. regardless of the formulation method used (Table 7). with small differences (Figure 7A). Results are presented in Figure 7. viable cell density decreased 1 log on the 3rd week.E.0 ˘ 1.5 ˘ 0. Characterization of batches of BCG-loaded “F14_Low” microparticles prepared with increasing concentrations of BCG.9 D II 1. BCG-loaded microparticles presented lower zeta potential values regardless of the used formulation method (Table 7). however. Formulation BCG Pasteur Particle Size.3ˆ107 21.0 ˘ 3. depending on the formulation method (Table 7). In comparison to plain microparticles.2 ˘ 1.4 I III 1.2 ˘ 0. Therefore.025% chitosan induced a decrease in BCG cell viability. as described in the Materials and Methods (Section 3.0 2.9% NaCl.0 ˘ 10. with a viable cell density decrease of 1–2 log on the third week.7ˆ106 22.3 ˘ 2. cell viability was further reduced.5–4 log on the 6th week. the microencapsulation procedures preserved BCG integrity and viability.6 1.5 mV to +14. Therefore. with zeta potential values from +10.0 ˘ 5.1 ˘ 0. Results showed a significant reduction of BCG cell viability for both BCG strains.4 ˘ 2.05). whereas Method (III) produced electronegatively charged microparticles (both plain and BCG-loaded).9 2.4 76.025% chitosan or in 0.5 ˘ 1.5 3.2 ˘ 0. we accept that BCG bacilli are sometimes microencapsulated and other times just adsorbed due to partial and irregular adsorption onto the microparticle surface.5) (µm) (mV) A II . Therefore.1 10.3ˆ106 28.2 ˘ 1.3 ˘ 3.2 ˘ 0.0 ˘ 0.

  The  The obtained  obtained results  results are  in  are in conformity with other studies where chitosan did not interfere with cell viability [31]. 25  μg/mL  is  25 µg/mL is about  about five  five times  times higher). incubation.Mar. BCG suspension in 0.9% NaCl BCG Pasteur / 0. 90 17 of 30 Mar.  (e.  presenting cells [55]. 25 µg/mL (p < 0.9% NaCl (angled lines). 12. BCG suspension in 0. Results  showed  Results showed no  significant  reduction no significant reduction  of  cellular  viability of cellular viability  after after  24 24  hh  incubation incubation  with with  chitosan‐suspended BCG and  chitosan-suspended BCG and BCG‐loaded  BCG-loaded microparticles.  (p and and < 0. agar agar (Middlebrook (Middlebrook  7H10 7H10  medium medium  supplemented  with  supplemented with OADC)  OADC) plates  plates inoculated  inoculated with  with bacteria  bacteria of  of both  strains  (Pasteur both strains (Pasteur  and  GFP)  that and GFP) that  presented  aa  number presented number  of of  colonies colonies  of of  statistical statistical  relevance relevance  were were used used to to calculate calculate the the CFU/mL.3 µg/mL 0. 25 μg/mL (p < 0. Drugs 2016.025% low molecular chitosan‐alginate microparticles (no fill). In Vitro Cell Viability (MTT Assay)  2.05).D. a  high  concentration  a high concentrationof of BCG‐loaded  BCG-loadedmicroparticles  microparticles(12. mean ± S.9% NaCl 6 BCG-GFP / 0.5  (12. 14. BCG suspension in 0. control. Results are expressed as  mean ˘ S. CFU/mL.00001). In Vitro Cell Viability (MTT Assay) The in vitro biocompatibility of the microparticles was evaluated with the MTT assay using a  The in vitro biocompatibility of the microparticles was evaluated with the MTT assay using aPMA‐differentiated  PMA-differentiatedTHP‐1 THP-1cell  cellline  line(Figure  (Figure 8).  by by multiplying  the  multiplying colony  forming the colony forming  units by the units  by the plating plating factor factor and and the the dilution dilution factor.5 μg/mL). although some formulations induced a slight decrease in cell viability (15%–20%).  conformity with other studies where chitosan did not interfere with cell viability [31].4.9% NaCl (angled lines).0077) and 6. none of the  BCG-loaded the BCG‐loaded  microparticles  microparticles may  may be be considered  considered as  cytotoxic  since  as cytotoxic since the  average  values the average values  were were  not not  significantly different  significantly from  the different from the control control group group at at tested tested concentrations.9% NaCl was was  used used  as as  control. BCG suspension in 0.025% CS 5 5 4 4 CFUs/ml (log) CFUs/ml (log) 3 3 2 2 1 1 0 0 0 3 6 9 12 15 0 3 6 9 12 15 Time (w eek) Time (w eek)   (B) 10 9 8 7 CFUs/ml (log) 6 5 4 3 2 1 0 week 0 week 3   Figure  7. except  except for  the  highest for the highest  concentrations concentrations  of of  chitosan‐suspended BCG. which  which is  is recommended  recommended as  as a  a model  model for  antigen  for antigen presenting cells [55].5 µg/mL (p = 0.g.  (B) (B)  BCG BCG  Pasteur Pasteur  viability following BCG  viability  following  BCG microencapsulation  microencapsulation in  in “F14_Low”  “F14_Low” chitosan-alginate microparticles (no fill).5 μg/mL (p = 0. Results are expressed as weight (horizontal lines).0077) and 6.  higher). although some formulations induced a slight decrease in cell viability (15%–20%).05).  2. 12. or BCG suspension in 0.D.3 μg/mL (p <  chitosan-suspended BCG.g. n = 3. Overall. or BCG suspension in 0. Drugs 2016. concentrations.00001).025% CS BCG-GFP / 0. none of  Overall.4.  Cell viability  Cell viability of  of BCG  (A)  After BCG (A) After  33 weeks  weeks of of incubation... namely. . n = 3. The The CFU/mL CFU/mL  provides an approximation of the cell density of the original culture. namely.. 14.  microparticles.025% low molecular weight chitosan  weight chitosan weight (horizontal lines). Figure 7. 90  17 of 30  (A) 6 BCG Pasteur / 0.  8).9% NaCl  provides an approximation of the cell density of the original culture.. factor.These  These concentrations  concentrations exceed  concentrations exceed concentrations  intended  intended for  for vaccination  vaccination assays  assays (e.  µg/mL).

(Paisley.05.  of 85%. New Lakes.56).  Franklin Franklin  Lakes.  ultra-purified water.s.  CFUs/mL). 3. 3. (structural  viscosity  (structural as  viscosity provided by suppliers).  by suppliers).  dark  grey—BCG‐GFP/0.  light grey—BCG‐GFP/0. were  a were gift  afrom  gift FMC  from BioPolymer A. (63%)  (63%) high G-content Manugel  LBA  (773  Manugel LBAmPa. Drugs 2016.  by the Columns:  MTT reduction. Preparation of Polymeric Microparticles  .  UK).S. Cell viability (% of control) = [A] test/[A] control ˆ 100. 20 °C) and undefined deacetylation degree. Materials and Methods 3. Norway). 20 C) and undefined deacetylation degree. were kindly supplied by Prof Elsa Anes Elsa  Anes  (FFUL). M/G  ratio  of  10 mg/mL.  100  mg/mL). medium viscosity (3000  Sodium alginate polymers of high viscosity (14. ** p < 0.5 25. all  ˝ structural viscosity (748 mPas. (Sandvika. from  Difco. (ATCC.01.1.001.  (3000 mPa. Collection (ATCC) Manassas. ** p < 0.025%LMW  LMW chitosan. UK). M/G ratio of 1. UK)UK)  (specifications  (specifications as  provided  as provided by  suppliers). 14.6 3.  (773 mPa.000 mPa.s. The molecular The  molecular  weights were weights  were  not Chitosan not verified. UK).  (218 mPa.01. dotted  white—BCG  Pasteur/F13_Medium dotted white—BCG Pasteur/F13_Medium  microparticles. were kindly supplied by Prof  for expression of Green Fluorescent Protein (rBCG-GFP) [76].s.000 mPa. 1% in acetic acid 1%. 8. 20 mg/mL). Norway). and  high  and G‐content  (65%–75%)  high G-content Protanal  (65%–75%) LF  10/60  Protanal (20–70 (20–70 LF 10/60 mPa. M/G ratio of 1. USA.0 Tested concentration (g/mL)   Figure  Figure 8. 14.05.56) from  Sigma‐Aldrich  were purchased from Sigma-Aldrich(Dorset.s. all purchased  purchased from from Sigma–Aldrich Sigma–Aldrich  (Dorset. verified. New Jersey. 2. and low viscosity (187 mPa.3 12. Sodium tripolyphosphate (TPP) and calcium chloride (CaCl FMC BioPolymer A. stock Chitosan  stock were solutions solutions  were inprepared  prepared 1% aceticin acid 1%  solution acetic  acid  in solution in ultra‐purified water.S.1 6.s. 90  18 of 30  140 * ** *** * Cell viability (% of control) 120 100 80 60 40 20 0 1. Statistical differences between the control group and formulations are reported as: *** p < 0.  (Dorset.  20  mg/mL).  3‐(4.025%  grey—BCG-GFP/0. 10 mg/mL. Materials and Methods  3. * p < 0. Statistical differences between the control group and formulations are reported as:  mean ˘ SD (n = 3).  Alginate  stock  solutions  (CaCl2 ) were purchased from Sigma-Aldrich (Dorset. bovis BCG Pasteur and rBCG‐GFP cultures were grown on Middlebrook’s  USA. bovis BCG Pasteur and rBCG-GFP cultures were grown on Middlebrook’s 7H9 broth 7H9 broth Medium supplemented with 5% (v/v) OADC (oleic acid. 1% in acetic acid 1%. albumin.  USA).Results  Resultsare  are expressed  expressed as  as mean ± SD (n = 3).  UK)  UK) (Dorset. Materials  3. dextrose and catalase supplement) supplement) at 37 °C/5% CO at 37 ˝ C/5% CO2 .s. Cell viability (% of control) = [A] test/[A] control × 100. bovis BCG Pasteur strain 1173 (ATCC 35734™) (American Type Culture Collection  (ATCC) Manassas.  microparticles. light  cells  with  culture  medium.  Jersey.s.56).  mPa. Both M.56)  were  ratio purchased  of 1.001.9%  NaCl. The BCG strains—M. Relative  cell  viability  Relative of  THP‐1  cell viability cell  line  of THP-1 measured  cell by  the  MTT  line measured reduction.  UK). * p < 0.s. Both M.Mar. 90 18 of 30 Mar.  VA. medium viscosity mPa.1. andand  chitosan  chitosan of of  highhigh  molecular  molecular weight (≈600  weight kDa) or («600 kDa) or  high  high structural viscosity (748 mPas. black—control  Columns: black—control cells with culture medium.  chitosan. Alginate stock solutions were prepared in were  prepared  in  ultra‐purified water.s. 20 mg/mL) (27 mPa.s. 100 mg/mL).  10  mg/mL). UK). BCG M. bovis bovis harboring BCG  harboring  a pMN437 a  pMN437  plasmid plasmid for expression of Green Fluorescent Protein (rBCG‐GFP) [76].  chitosan  (<150 kDa) and deacetylation degree of 92%.VA.  bromide (MTT).s.  The  THP1  cells  (ATCC  The THP1 cells (ATCC TIB-202™) TIB‐202™)  a  human  a human monocyte  monocyte cell cell  line line  was was  obtained  obtained from from  (ATCC.  USA)and  USA)and a  recombinant  a recombinant M. The(FFUL). (Sandvika. USA). dark grey—BCG-GFP/0. dextrose and catalase  Medium supplemented with 5% (v/v) OADC (oleic acid.9% NaCl.5-dimethyl-2-thiazolyl)-2. Phorbol UK). 3-(4. and low viscosity (187 mPa. 20 mg/mL) (27 mPa. dotted  dotted grey—BCG  Pasteur/F13_High  microparticles  grey—BCG Pasteur/F13_High microparticles (1  (1 ×  ˆ 108  108 CFUs/mL).5-diphenyl-2H-tetrazolium bromide  (MTT).5‐diphenyl‐2H‐tetrazolium  (PMA). Other tested sodium alginates include: low G-content (40%) Keltone (218  LVCRmPa. Most  experiments were Most experiments were  performed  performed using  using solutions  solutions containing  containing chitosan  chitosan of  low‐molecular  of low-molecular weight weight  (<150  kDa)  and  deacetylation  degree  of  92%.  (DMSO) were all from Sigma-Aldrich (Dorset. chitosan of medium molecular weight of  medium  molecular  weight  (<450 (<450  kDa) kDa) and  deacetylation  and deacetylation degreedegree  of 85%.2. high  G‐content  20 mg/mL). dimethylsulfoxide dimethylsulfoxide (DMSO) were all from Sigma‐Aldrich (Dorset.  *** p < 0. Drugs 2016.  Phorbol  myristate acetate myristate acetate  (PMA).  ultra-purified water. Sodium tripolyphosphate (TPP) and calcium chloride 2)  were  purchased  from  Sigma‐Aldrich  (Dorset. M/G 1. Materials Sodium alginate polymers of high viscosity (14.s. Other tested sodium alginates include: low G‐content (40%) Keltone LVCR  as provided by suppliers). which which 10 mg/mL).s.  The  bacterial bacterial  cell culturecell  culture  reagents reagents  were were from purchased purchased  Difco. bovis BCG Pasteur strain 1173 (ATCC 35734™) (American Type Culture  The BCG strains—M.s. 20 mg/mL).5‐dimethyl‐2‐thiazolyl)‐2. 20 mg/mL. 20 mg/mL.  All cell All animal animal  cell  culture culture were reagents reagents  were from purchased purchased  from  Invitrogen Invitrogen  (Paisley. albumin.

120–150  chitosan minutes.  addition (adapted from [33]). by  means  by means of  ionic  of ionic interaction  interaction betweenbetween  positively  positively chargedcharged  amine  amine groups groups of chitosan and negatively charged carboxyl groups of alginate.02:1 to 1:1 (w:w).10.   Figure 9.  ratios ranged from 0. Then. Briefly. a 7. allowing  allowing sodium  sodium alginate  alginate solution  solution to  with to react react calcium with  calcium  chloridechloride  prior  to addition.  respectively  (alginate: (alginate:  chitosan mass ratio chitosan  4. Drugs 2016.5 mL aliquot of 18 mM calcium chloride solution was added drop  (Figure 9). and stirred for 60  beaker containing 117. 90 19 of 30 Mar. Alginate and chitosan were dissolved in ultra-purified water. Amass  ratio  colloidal 4. and  0. Briefly.9. followed by dropwise addition addition  of 1. prior to TPP addition (Figure  of chitosan and negatively charged carboxyl groups of alginate. 14.0 1. prior to TPP addition (Figure 10). 10).5 mg/mL andof  0. Alginate and chitosan were dissolved in ultra‐purified water.1 to 5. giving a final alginate and chitosan  drop wise into the pre-gel and stirred over 90 min.0 mL of 1. to provide an alginate pre-gel.  formed upon polycationic chitosan addition.0 mg/mL chitosan were added drop wise into a beaker containing volume volume ranging from 0. 25.  A  dispersion colloidal dispersion formed upon polycationic chitosan addition.0 mg/mL mL  of  1. giving a final alginate and chitosan concentration of concentration  0.2. byby  pre-gel.0 mg/mL of sodium alginate solution.0 mL of a 0.23:1). Briefly. polymeric microparticles were prepared by allowing chitosan and sodium  alginate  sodium alginate toto  polymerize.02:1 to 1:1 (w:w). a 5.6 mg/mL sodium alginate solution. followed by dropwise  ranging from 0. by high speed stirring at room temperature and without organic solvents—Methods (I) and (II). 25. and stirred for 60 min under min under 600 rpm.0 mL aliquot of 1.1  mg/mL.Mar. Then. 14. polymeric microparticles were prepared via formation of an alginate ionotropic pre‐gel.  polymerize.0  TPPmg/mL  TPP  under stirring under high-speed high‐speed  at 600stirring  rpm forat  600  rpm  120–150 for Alginate: min.0 mL of a 0. polymeric microparticles were prepared by allowing chitosan and  In formulation Method (II). In Method (I).7 mg/mL chitosan solution  600 rpm.5 mL of a 0. prior to chitosan chitosan  addition.  Alginate: chitosan ratios ranged from 0.0 mL aliquot of 1. .5  mg/mL respectively mg/mL. to provide an alginate pre‐gel.5 mL of a 0. Preparation of Polymeric Microparticles Polymeric microparticles Polymeric microparticles were  initially prepared were initially prepared using  modifications of using modifications of previously previously described described  ionic cross‐linking methods [30–34].1 to 5. a 5.  Figure Microparticles  Microparticles formation  formation by  alginate  by alginate ionotropic  ionotropic pre‐gelation  pre-gelation with  with CaCl CaCl2  followed  2 followed by  by chitosan chitosan addition (adapted from [33]). In formulation Method (II).5 mL aliquot of 18 mM calcium chloride solution was added drop wise into awise into a beaker containing 117.0 mg/mL chitosan were added drop wise into a beaker containing  Briefly. Drugs 2016. by high speed stirring at room temperature and without organic  ionic cross-linking methods [30–34].0 mg/mL of sodium alginate solution.0 mL of 1.  solvents—Methods (I) and (II). polymeric microparticles were prepared via formation of an alginate ionotropic  In Method (I). (Figure 9). a 7.0 mL ofof 1. 90  19 of 30  3.7 mg/mL chitosan solution was added was added drop wise into the pre‐gel and stirred over 90 min.23:1).6 mg/mL sodium alginate solution.

06:1  F4 0.068 0.065  0. Therefore.017 ** 0.009 0.042 0.005  0.20:1  0.017 **  1. Values for the investigated variables during formulation studies.036  with CaCl2 followed0.010 *  0. 14.026 **  0.069 0. 90 20 of 30 Mar.Mar.40:1  F12 F14 0.12:1 F6 F7 0. and TPP.068  0. high. obtained via alginate 0. were used and their volume was kept constant (5.10:1  0. Table 8.065 0.60:1 F11 F13 0.018 **  by chitosan addition—Method (I).042  (II).026 ** 0.10:1 F5 F6 0.018 ** 0. Drugs 2016.027 **  0. on parameters such as particle size and zeta potential.00:1  2 .036 0. 90  20 of 30    Figure 10.070  0.026 ** 0.063  0.028 **  0.020 **  1.022 0.40:1 F10 F12 0.007  0.070 0.025 ** 0.022 ** 0.008  0.050  0. medium and low and low molecular weight chitosan.067 0.025 **  0. Therefore.005 0. and respectively). high.34]).  mL.009  0.23:1 F1 F1 0.004 0.34]).008 0.069  0.025 ** 0.  Chitosan (CS)  Chitosan (CS) Alginate (ALG) Alginate (ALG) CaCl*2* or TPP ** CaCl ALG:CS Mass Ratio  Formulation  2 or TPP ** ALG:CS Mass Ratio Formulation (% w/v)  (% w/v)  (% w/v)  (w/w)  (% w/v) (% w/v) (% w/v) (w/w) F0 F0 0.027 ** 0. medium  mass ratio.022 **  0.050  0.  used and  medium  and low  theirviscosity  volumealginate  was kept constant was  used  in (5.011  0.027 **  0.066 0.063  0.02:1 F2  F2 0.026 **  0. 14.063 0. Microparticles formation by chitosan gel matrix formation with sodium alginate followed  Figure 10.011 0. Microparticles formation by chitosan gel matrix formation with sodium alginate followed by TPP addition (adapted from [32.050 0.80:1  0.028 **  0.18:1 F8  0. Values for the investigated variables during formulation studies. Drugs 2016.050 0. such as different pH values and polymers polymers mass ratio.0 volumes.030 0.030  0.  such  as  different  pH  values  and  impact of the key components of the polyelectrolyte matrix.16:1 0.027 **  0.04:1  F3 F3 0.60:1  F0—ALG/CS F13  microparticles.042  ** TPP.013  0.80:1 0.010  0. respectively).007 0.  chitosan.028 ** 0.12:1  0. Table 8.004  0.026 ** 0. 0.001 0.020 ** 0.20:1 F9 F11 0.010 0.003  0.04:1 0.068  0.025 **  0.010 0.08:1  0.056 0.00:1 0.010  0.064  0. while  were high. obtained via chitosan precipitation with TPP followed F14  with alginate—Method by gelation 0.0  mL.066  0.18:1  0.08:1 F4 F5 0. while high.23:1  4.056  0.028 ** 0. alginate and sodium tripolyphosphate.0 mL  molecular weight and  2. according to previously  described  method  [32]  with  modifications  (Table  8).026 **  0.003 0.022  0.06:1 0.068 0.064 0. medium and low viscosity alginate was used in increasing volumes.  TPP.16:1  F10 0.013 0.010 * 4. according to a  appropriate concentration range for chitosan. * CaCl 0. Preliminary  experiments  with  three  replicates  were  designed  in  order  to  investigate  the  Preliminary experiments with three replicates were designed in order to investigate the appropriate concentration range for chitosan.14:1  F9 0.02:1  0.067  0. on parameters such as particle size and zeta potential.045  ionotropic pre-gelation 0.042 0.027 ** 0.  by TPP addition (adapted from [32.063 0.001  0.045 0. alginate and sodium tripolyphosphate. F1–F14—CS/ALG microparticles. The purpose was to identify the impact  of  the  key  components  of  the  polyelectrolyte  matrix.027 ** 0.  The  purpose  was  to  identify  the  a previously described method [32] with modifications (Table 8).14:1 F7  F8 0.0 mL and increasing  2.

also added drop wise. microparticles were prepared with sodium alginate of different quality specifications. equipped with a 3 mm microtip probe.0  of 1.  Microparticle  of aggregates) preparation  and microparticles was  in  characteristics. %).5 mL of 0.75 mL of 0. in accordance with chloride were added to 11.  with  such as size distribution and surface charge.  Microparticles  Microparticles formation  formation by  chitosan  by chitosan precipitation  precipitation with  with TPP TPP  followed  followed by addition. USA) were assessed as potential alternatives to high speed microparticle  stirring suspensions  to prepare (general  microparticles.1–5. polymeric microparticles were prepared by inducing the pre‐gelation of chitosan with TPP. BRANSON stirring to prepare microparticles. followed  polymeric microparticles were prepared by inducing the pre-gelation of chitosan with TPP.3 . and either homogenized for 3 min  previously described formulation Method (I).0mg/mL  mg/mLsodium  sodiumalginate solution. IKA-Labortechnik.  TPP. 5. Mar. viscosity and guluronic acid monomers content (G-content. prior to 2. The results The results obtained obtained during during formulation formulation optimization optimization studies studies led led us us towards towards the the rejection rejection of of  Method (I) and the development of a different preparation method—Method (III).75 mL of 0. 14. followed by addition of 1 mL of 2.  Briefly.0 mL mLof 1. 14.0 mg/mL of sodium alginate. charge.400  rpm.  21 of 30 High  shear  homogenization  (ultra‐turrax  T10basic  at  11. a wide range of chitosan.  Method (I) and the development of a different preparation method—Method (III). and either homogenized for 3 min in ultra-turrax or sonicated for 3 min at 20% output intensity. 0. CT.75 mL of 18 mM calcium  described formulation Method (I) and Method (II).75 mL of 18 mM calcium chloride were added to in ultra‐turrax or sonicated for 3 min at 20% output intensity.0  mg/mL  for Method (II).0 mL of 1. formation charge. Ultrasonics Corporation. with modifications.0  as described. Microparticles were formed by the dropwise addition of a 1.  by alginate coating (Figure 11).0 mL of 1.400 rpm.1 to  to 5. best  experimental  conditions  to  obtain  microparticles  of  intended  size  distribution  and  surface  In these studies.and sodium alginate-solution pH value was assessed (pH from 3.  microparticles  were  prepared  with  sodium  alginate  of  different  quality  namely.  IKA‐Labortechnik. and the influence of pH value variations in microparticle size distribution and surface charge was determined.6 mg/mL sodium alginate. alginate and chitosan were dissolved in ultra‐purified water. Danbury.0 mL aliquot of 2. CT. AsmL  of  1. and subsequent homogenization or sonication as described.6 mg/mL sodium alginate.0 mg/mL TPP. Briefly.7 mg/mL chitosan addition. 0. As for Method (II).   Figure 11.0 mg/mL TPP into a  Microparticles were formed by the dropwise addition of a 1.1–5. in accordance with previously described formulation Method (I).0of  mLsodium  alginate. Briefly. Briefly.1  ranging mL mL from 0. alginate and chitosan were dissolved in ultra-purified water. 5.0 mg/mL chitosan solution. In Method (III).7 mg/mL chitosan  11. Microparticle preparation was in accordance with above modifications. The techniques were evaluated considering physical stability of  Ultrasonics Corporation. obtained via chitosan precipitation  with TPP followed by gelation with alginate—Method (II). under high‐speed stirring at 600 rpm for 120–150 minutes. prior to 2. accordance  with  above  described  formulation  Method  (I)  and  Method  (II). and subsequent 1. Staufen. equipped with a 3 mm microtip probe.  of 1.0 mg/mL TPP into beaker containing 5. followed by alginate coating (Figure 11).  formation  The techniques were evaluatedof  aggregates)  and  microparticles  considering physical stability of characteristics. Figure 11.0  mg/mL  chitosan  homogenization were  added  or sonication to  0. also added  drop  alginate solution. BRANSON  High shear homogenization (ultra-turrax T10basic at 11.0 mL of 1. under high-speed stirring at 600 rpm for 120–150 min.Mar. USA) were assessed as potential alternatives to high speed  Germany) and sonication (Branson Sonifier 250.0 5.0 mL aliquot of 2. In Method (III).0 mg/mL followed  chitosan by  were addition of 1 mL of 2. by alginate alginate  addition. During pH value studies.  obtained  via  alginate  ionotropic  pre‐gelation  with  CaCl2  followed  by  chitosan addition—Method (I). 90  * CaCl2 ** TPP. and either homogenized for 3 min in ultra‐turrax or sonicated  added to 0. Drugs 2016. and either homogenized for 3 min in ultra-turrax or sonicated addition.0 mL of  for 3 min at 20% output intensity.  In  these  studies. followed by polyanionic cross linking volumes  with ranging  volumes from 0. followed by polyanionic cross linking with  a beaker containing 5. aspect. 90  21 of 30  F0—ALG/CS  microparticles.  microparticle such  as  size  suspensions distribution  (general and  surface  aspect.  Germany) and sonication (Branson Sonifier 250. Danbury.  Staufen. F1–F14—CS/ALG microparticles.  Additional characterization studies were conducted with Methods (II) and (III) to identify the Additional characterization studies were conducted with Methods (II) and (III) to identify the  best experimental conditions to obtain microparticles of intended size distribution and surface charge.5 mL of 0.0 mg/mL for 3 min at 20% output intensity.  wise.0 mg/mL chitosan solution. Drugs 2016.

followed by drop wise addition of 4.0 mg/mL chitosan solution. 3.9% (range. bovis BCG [77]). size distribution and surface charge were determined. Mycobacteria were submitted to heat inactivation. This was later confirmed by methylene blue staining and haematocytometer count (for BCG Pasteur) and CFU counts after bacterial suspension plating. Modifications to the formulation methods were introduced along the optimization process accordingly to the conclusions yielded by the obtained results during preliminary studies. 3. Microencapsulation of BCG BCG-loaded microparticles were prepared by addition of 1.1%. by submersion of 15 mL falcon containing bacterial suspensions in a water bath preheated and maintained at 80 ˝ C for 15 min [82]. and to perform the further characterisation studies in safety. the OD of the suspension at λ = 600 nm was adjusted to 0.6). For bacteria remaining in clumps.4 and re-suspended in appropriate medium. some preliminary studies using two concentrations (5% and 10% w/v) of three different cryoprotectors (sucrose. a colony-forming units (CFUs) assay was used to count bacteria of both strains (Pasteur and GFP) able to produce colonies in agar Middlebrook 7H10 medium supplemented with OADC (widely used to cultivate and access the CFUs in the case of slow growers such as M. BCG Studies 3. The suspension was kept on the bench for 5 min.0 to 5.25 mg/mL . while alginate (Protanal™ LF 10/60) concentration was kept constant at 0. 3. 1.4. 1–2 ˆ 108 CFUs/mL) to 5. as this can vary immensely as a function of the growth medium composition of mycobacteria.Mar. 3.3.0 mL of 1.1 (we assumed that 0.3.2. Inactivation studies of both strains were also conducted to assess the effect of viability loss in BCG surface characteristics. 90 22 of 30 to 7. monodispersed bacteria were suspended in different concentrations of chitosan and encapsulated or adsorbed into different formulations of microparticles. in order to get individualized bacilli. to allow the decantation of the large clumps of bacteria. Regarding future lyophilisation of microparticles. and trehalose) were also conducted. and also due to a potential growth inhibition effect of chitosan [77–81]. Next.0 mg/mL sodium alginate solution to the mixture.0 mg/mL TPP was added drop wise to chitosan-suspended BCG. washed twice in sterile 10 mM PBS pH 7. aliquots of BCG suspended in 0.3.0 mL of 2. Briefly. in order to more accurately determine the total viable number of bacteria. In the absence of clumps. Clumps of bacteria were removed by ultrasonic treatment of bacteria suspensions in an ultrasonic water bath for 15 min. BCG Cell Viability In order to assess BCG viability.1 OD600 corresponds to 1 ˆ 107 bacteria per mL [76]). The efficacy of inactivation methods was determined by viability checks. Surface Charge Characterization In order to modify the physicochemical properties of BCG. These studies were performed for both BCG Pasteur and rBCG-GFP strains. 14. BCG Single Cell Suspension Bacterial cultures in exponential growth phase (after 7 days) were pelleted at 4000 rpm (1559ˆ g) at 4 ˝ C for 15 min. as follows: 100 µL of the heat killed suspension was used to inoculate each of two plates with solid agar Middlebrook 7H10 medium supplemented with 5% OADC and incubated at 37 ˝ C in 5% CO2 atmosphere for 3 weeks. the complete volume of the suspension was collected and pressed through a 21 gauge needle against the syringe tube wall for 10 times.45 mg/mL of chitosan. Drugs 2016.3.0 mL of 1. tuberculosis and M.1. Final concentrations of prepared microparticles ranged from 6 to 8 log10 CFUs/mL and from 0.0 mL of whole live attenuated BCG bacillus monodispersed in NaCl 0.42 to 0.3. Then.3. Single cell suspension was confirmed by phase contrast microscopy or in a fluorescence microscope (for rBCG-GFP). glucose.

between 4000 and 500 cm´1 [83]. each of them measured three times. UK). Results were expressed in terms of mean diameter and span (Span = d (0. Bethesda. The samples were maintained at 4 ˝ C for approximately four months (15 weeks). CA.3. After three weeks of incubation at 37 ˝ C and 5% CO2 . The mean values were obtained from the analysis of three different batches. USA).Mar. The FT-IR measurements were made directly in the dried microparticles. Characterization of Microparticles 3. 3.4. 3. All spectra were recorded at room temperature at the resolution of 4 cm´1 and 50-times scanning. 3. Total cells ´ Free cells Encapsulation efficiency p%q “ ˆ 100 (2) Total cells .4. Kyoto. Encapsulation Efficiency Encapsulation efficiency (EE. respectively (Malvern Instruments. Drugs 2016.4. Size Distribution. and all powder raw materials namely chitosan and alginate. using a Mastersizer 2000 and a Malvern Zetasizer. 1.4.2. Worcestershire.5)). colonies were counted to determine CFUs. For particle size analysis.4. Production Yield The production yield (YP) (Equation (1)) of the microparticles was determined using an indirect method based on the quantification of the chitosan concentration initially used in the formulation. Japan) spectrophotometer. 90 23 of 30 chitosan of medium MW and BCG-loaded chitosan-alginate microparticles were seeded in appropriate inoculation medium to determine the effects of processing conditions on cell viability.4. Norwalk. rCSs total ´ rCSs supernantant CS yield p%q “ ˆ 100 (1) rCSs total 3. Size distribution is characterized using the d0. which were previously lyophilised. The ImageJ software.9) ´ d (0.44p version (National Institutes of Health. For the determination of the electrophoretic mobility. samples were diluted with filtered purified water.1)/d (0.1. MA. and plates were inoculated with samples for cell count at regular time points. The encapsulation efficiency is expressed as the percentage of BCG entrapped/adsorbed in microparticles reported to initial amount of cells in suspension (Equation (2)). The method of quantification is based on a colorimetric reaction between amine groups of chitosan and the dye Cibacron brilliant red 3B-A [30]. each sample was diluted with filtered purified water to the appropriate concentration to yield 10% obscurity limit. %) was determined by cell count number using a haemocytometer (Neubauer chamber Bürker). 14. Surface Charge and Morphology The microparticles were assessed according to size distribution and surface charge (zeta potential). Fourier Transform Infrared Spectroscopy (FT-IR) Analysis Preliminary information on chemical nature of the chitosan-alginate microparticles was collected using FT-IR analysis in an IRAffinity-1 (Shimadzu Corporation. gently mixed with approximately 300 mg of micronized KBr powder and compressed into discs at a force of 10 kN for 1 min using a manual tablet presser (Perkin Elmer. and that found in the supernatant of the final microparticle suspension as previously published [29]. by laser diffraction and electrophoretic mobility. Each analysis was carried out in triplicate at 25 ˝ C. Morphological examination of microparticles was performed by microscopy.5 parameter (diameter for which 50% of the distribution falls below) and the span parameter (width of particle size distribution). USA) was used to perform image analysis.

such as the properties of the used polymers. namely surface charge. where Abs test is the absorbance value obtained for cells treated with samples. Each sample concentration was tested in triplicate in a single experiment. polymer/polymer and polymer/complexation agent mass ratios. CA. penicillin and streptomycin. It was possible to develop a reproducible method for microencapsulation of whole . encapsulation efficiency and yield of preparation. 5.05 was considered to be significant.6. THP-1 cells (grown in RPMI 1640 supplemented with 10% FBS. 4.02 (GraphPad Software. Männedorf. 14. Briefly. type. it was possible to optimize the preparation method for BCG-loaded chitosan-alginate microparticles with reproducible size distribution. Statistical Analysis Data were subjected to ANOVA for analysis of statistical significance. It was possible to observe that.73]. Switzerland). After 15 min at room temperature. speed and duration of homogenization. and rBCG-GFP). as well as two strains of Mycobacterium bovis BCG (BCG Pasteur. for chitosan-alginate microparticles.Mar. After the incubation time. These parameters are influenced by several experimental conditions. which was repeated at least 3 times. The number of variables that could be optimized was reduced throughout the formulation development. Abs test Cell viability p% of Controlq “ ˆ 100 (3) Abs control 3. results are expressed as mean values ˘ standard deviation (SD).) were seeded onto 96 well cultures dishes at a density of 5 ˆ 105 cells/mL and treated for 72 h with 20 nM PMA in order to differentiate into macrophage. Particle size and size distribution uniformity were considered to be critical aspects throughout the formulation studies. size distribution was mainly influenced by the molecular weight of the used polymers and by the type of polymer blends. it was possible to tune BCG physicochemical properties. were used. Cells were then incubated for 72 h at 37 ˝ C with different concentrations of CS and ALG solutions and BCG loaded and empty formulations. On the contrary. Additionally. the optimization of the preparation method relied on the identification of the best polymeric compositions and identification of the crucial steps in the ionic gelation methods that were determinant for particle size distribution and surface charge. USA). Essentially. culture medium was replaced with culture medium containing 0. the absorbance of the extracted solution was measured at 570 nm in a microplate reader (Infinite M200. Drugs 2016. Controls consisted of cells incubated with only culture medium. The medium was removed after 3 h and the intracellular formazan crystals were solubilised and extracted with dimethylsulfoxide (DMSO). The analysis was carried out using GraphPad Prism v. antigen type (whole live bacteria represent additional challenges regarding cell viability maintenance during formulation). and the relationship between the pH values of the different polymers. and medium exchanged and incubated for one more day. In this study. clinically available vaccine. 90 24 of 30 3. The percentage of cell viability was determined for each concentration of tested sample according to Equation (3). La Jolla. and Abs control is the absorbance value obtained for cells incubated with culture medium. Conclusions During these formulation studies. By simply suspending BCG in chitosan. biodegradable and biocompatible polymers (chitosan and sodium alginate). Unless stated otherwise. and a p value of <0.5 mg/mL of MTT and incubated for 3 h at 37 ˝ C. the encapsulation of monodispersed whole live BCG bacilli into microparticles was of paramount importance since it could directly influence BCG cell viability and particle size and surface charge. Tecan.5. In Vitro Cell Viability (MTT Assay) Animal cell viability was assessed using general cell viability endpoint MTT as previously described with some modification [30. particle surface charge was mainly influenced by polymer to polymer mass ratio due to the possibility of particle aggregation.

and A. The microencapsulation of BCG had no considerable effect on particles key features (i. In conclusion. live attenuated. L.G.D.E.ULisboa for financial support (UID/DTP/04138/2013) from Fundação para a Ciência e Tecnologia (FCT). This was clearly shown with the empty particles (Tables 1. due to the negative surface charge of BCG bacilli. L.M. cell-based particulate delivery system was developed for mucosal immunization purposes. for access to the laboratory as well for all advice and practical support. Regarding particle surface charge. the concentrations of BCG used.J.A. Conflicts of Interest: The authors declare no conflict of interest.G. as follows: Method (I) produced negatively charged particles. morphology). L. L.e. depending on the polymer mass ratio (Figure 2) or polymer specifications such as alginate G-content (Figure 3).C. L. As expected. 90 25 of 30 live bacteria using only mild conditions. and L.A. and stoichiometric predominant. size distribution. it was possible to demonstrate that the addition order of the polymers was crucial to obtaining microparticles of either electronegative or electropositive surface charge. Author Contributions: L.G.. However.M. Nuno Carmo) by supplying strains (Pasteur and GFP). probably due to interference with the polymer arrangement. The authors thank iMed. Drugs 2016.D.. Acknowledgments: The authors would like to thanks to Elsa Anes (FFUL) and her team (David Pires. Portugal. BCG encapsulation led to minor modifications of the net charge at the particle surface (as depicted in Table 7).Mar. to a minor extent. wrote the paper.M. Abbreviations The following abbreviations are used in this manuscript: ALG Alginate APCs Antigen presenting cells BCG Bacillus Calmette-Guérin CFU Colony-forming unit CS Chitosan DMSO Dimethylsulfoxide E. Portugal and Escola Superior de Tecnologia da Saúde de Lisboa for financial support from merit scholarship for Ph. Encapsulation efficiency FT-IR Fourier transform infrared spectroscopy G Guluronate GFP Green fluorescence protein HMW High molecular weight HV High viscosity LMW Low molecular weight LV Low viscosity M Mannuronate .A. conceived and designed the experiments. Method (II) allowed the preparation of positively to negatively charged particles. surface charge. while maintaining cell viability and assuring the biocompatibility of the developed microparticulate delivery system.G. Further characterization of these formulations in terms of in vitro cellular interaction with macrophages and in vivo study following intranasal immunization in mice is ongoing. with good production yield and encapsulation efficiency.D. as alginate probably coated previously formed chitosan particles.C.J.A. performed the experiments. and A. proved to be crucial in achieving high encapsulation efficiency values. the formulation method and. a whole. and Method (III) allowed the preparation of negatively charged particles.D. 3 and 4). (ESTeSL-IPL/CGD/2012) granted by Caixa Geral de Depósitos. analyzed the data..A.D. 14. alginate matrix).C. as chitosan droplets were imprisoned in a previously formed. through ionic cross-linking.M.

. Atuah. M. Bramwell. M. [CrossRef] [PubMed] 2..R.E... 6. Immunity 2010. Synergistic effect of bacillus Calmette Guerin and a Tuberculosis subunit vaccine in cationic liposomes: Increased immunogenicity and protection.Mar.C..O. 3721–3730.M. Tabassi.E. S.O. [CrossRef] [PubMed] 6. Almeida. Kaufmann.T. Immunol. Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery.H. 4595–4601. Release 2010. Annu. 2002. M. I. S. Somavarapu.. [CrossRef] [PubMed] 3. D. Release 2010. Newport. J. Jaafari. M. H. 919–925. Bouwstra. P. pp. K. Cell-mediated immune responses in tuberculosis. S. Y. T. W. W. D. K. 207–226.A. O’Hagan. Billeskov. Alpar. J. Pharm. 393–422. [CrossRef] [PubMed] 5.O.. Vaccine 1998. 27. 90 26 of 30 MMW Medium molecular weight MV Medium viscosity MTT 3-(4.. M... Singh.. V. J. J.E. Dual role of CpG as immune modulator and physical crosslinker in ovalbumin loaded N-trimethyl chitosan (TMC) nanoparticles for nasal vaccination. Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN. [CrossRef] [PubMed] 14. Whittle. M. et al. Aaby. Henriksen-Lacey. Immunol.. Envisioning future strategies for vaccination against tuberculosis. Int.M. Rev. Rev. Vekemans. 2006. Harwood Academic Publishers: Reading. P.. Ota. Antigen Delivery Systems—Imunological and Technical Issues.J. 699–704. Kidd. Susanna.W. M. 698–707. 2007.. J. Fielding. Alpar. 19. Immunol. Immunol. Salgame. 168. M. Res. J... Vaccine 2007. dextrose and catalase supplement OD Optical density PBS Phosphate-buffered saline solution PLGA Poly(D. Intra nasal administration of poly-lactic acid microsphere co-encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse.A. Taghikhani. 567–577.O... Johansen. E. Ajdary. 2002. 57. 14. Nat. 2007. K. Oral administration of BCG encapsulated in alginate microspheres induces strong Th1 response in BALB/c mice.. 2006. [CrossRef] [PubMed] 7. Kaufmann. 715–728. Control.5-diphenyl-2H-tetrazolium bromide NALT Nasal associated lymphoid tissue ND Not determined OADC Oleic acid.. Jiskoot. Future vaccination strategies against tuberculosis: Thinking outside the box. 147. Dietrich.D. 33.L-lactide-co-glycolide) PLA Poly(L-lactide) PMA Phorbol myristate acetate rBCG Recombinant BCG THP-1 human monocyte cell line TPP Tripolyphosphate US Ultrasonication UT Ultraturrax YP Yield of production References 1. Spiers. .N.. Administration routes affect the quality of immune responses: A cross-sectional evaluation of particulate antigen-delivery systems. 319. Pharm. H. 148.J. 1997.. [CrossRef] [PubMed] 13. 117–121. [CrossRef] [PubMed] 9. Lambert.J. T. Clin.. Slütter. albumin.. [CrossRef] [PubMed] 4. P. Control. 2009. J. Tafaghodi. Cooper.. 25. B. G. Kündig. Mohanan. H.... [CrossRef] [PubMed] 8. P. Jiskoot.D. [CrossRef] 11. 2005. Bhatt. Perrie. Dobakhti. 178. 16. E. J.. Slütter. 10. Sharp. A.. Doherty. Rev. A. 411–430. R. Influence of Mycobacterium bovis bacillus Calmette-Guérin on antibody and cytokine responses to human neonatal vaccination. Immunol. F.M. B. 347–362. Alpar..H. Host innate immune response to Mycobacterium tuberculosis. S. [CrossRef] [PubMed] 12..S. Gander. J. H... 27. Drugs 2016. 342–349. S. M.5-dimethyl-2-thiazolyl)-2. B. Sanneh.. J. Eyles. Adv. P. Williamson. Recent advances in vaccine adjuvants. Andersen. UK.. 37–43. Drug Deliv.H.

A... Gonçalves...D.S. M.D. Development and characterization of a new plasmid delivery system based on chitosan-sodium deoxycholate nanoparticles. Polym.J. J. W. N. Vaccine 2012.. Sarmento. Sci.T. [CrossRef] [PubMed] 27.. Drug Deliv. C.. J. 45.L. Grabnar. Chesko. [CrossRef] 20.M. J. 3657–3666. 1431–1436. L. 2012.J. J. Rev. 298. Rev. Jilek. M. 2014..W.. 59–71. Gonçalves. Remuñán-López. Release 2006.. [CrossRef] [PubMed] 25.. [CrossRef] . J... R. Biomaterials 2010. Gonçalves. 7. A. C. Tang. J. 21–42. Drug Target. J. Verheul. 1–7.M. P. [CrossRef] 23. 76.. p. 2006. S. Coombes.. 31. L. Sci. M. Douglas.D. 2008. Sundblad. Pharm... 51. J. antigen loaded N-trimethyl chitosan nanoparticles for nasal and intradermal vaccination: Adjuvant. J. Frokjaer. 1997. S. 6551–6558. 323–335.. 879–891.K. Ferreira. 2001. Veiga. 45.R. Pharm. 16. Remuñán-López. Microencapsul. Almeida. H. K. Eur.. 28. [CrossRef] [PubMed] 26. Alpar. Gonçalves.J... S. C. Adv. Portugal. Improving vaccine delivery using novel adjuvant systems.J. [CrossRef] [PubMed] 21. C.M. L.. 90 27 of 30 15. M. P. 14. C. In Proceedings of the 2012 IEEE 2nd Portuguese Meeting in BioEngineering. A. Almeida. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. Ugozzoli. Alpar. 475–481. Tabrizian. Yeh.L. Vaccin. L. 2011.. M. 315–322. Nasal delivery of vaccines. 32. Bal. Alonso. M.J. S... J. IEEE EMBS Portuguese Chapter. Biomater. 2005. A. A. S. D. 1995.C. Does phagocytosis activity of dendritic cells measure up with macrophages? J.. 451–458. 2833–2841. Hum. Vajdy. Hu. Chitosan and Chitosan-Ethylene Oxide-Propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines.. O’Hagan. R.. L. [CrossRef] [PubMed] 31. Nanotechnol.I.G. H. Int. F. Effect of experimental parameters on the formation of alginate-chitosan nanoparticles and evaluation of their potential application as DNA carrier.C. Cadete.D. Calvo. J.and site-dependent immunogenicity in mice. Veiga.. Evaluation of particle uptake in human blood monocyte-derived cells in vitro. L. He. Adv. B. Vila-Jato.. Encapsulation of the immune potentiators MPL and RC529 in PLG microparticles enhances their potency. Pichichero. Calado. J. Kristl. [CrossRef] [PubMed] 17. 4. Characterization of insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation through DSC and FTIR studies. [CrossRef] [PubMed] 22. Rothen-Rutishauser. Aspects of the design and delivery of microparticles for vaccine applications. Almeida. [CrossRef] [PubMed] 19. I.. Drug Target. Yin.J. 1. L. 79–81. Pharm. Ribeiro. Insulin-loaded nanoparticles are prepared by alginate ionotropic pre-gelation followed by chitosan polyelectrolyte complexation.L. [CrossRef] 29. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model.R. Lopes. R. L. Almeida. F. C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. 2007.M. 3.M. Thomas.. L. Approaches to tuberculosis mucosal vaccine development using nanoparticles and microparticles: A review. Y. 455–467. P.. Kazzaz. Sarmento. [CrossRef] 28. 23–25 February 2012.D. Davis. Jenkins. Corvo. Ribeiro. L.. 14. Neufeld.. Adjuvanted.. Figueiredo.O. B. 43–56. Microparticles for intranasal immunization. Hagan.J. A. Biomed. D. [CrossRef] [PubMed] 30. Pandit. Davis. Ferreira. S.. 127–141. Florindo. [CrossRef] [PubMed] 33. Figueiredo.O. Yin. Soenawan. 51. Carbohydr. E. M. Slütter. Nanosci. Eur.J. Figueiredo. [CrossRef] [PubMed] 18..T. J. Pharm. Nasal vaccines. J.O. 125–132. 2295–2316.. Calado. A.. D. [CrossRef] [PubMed] 24. 1997.. [CrossRef] [PubMed] 16. A.M. J.C. 1996. L.J.G.A.L. A. Thiele. Protein and DNA nanoparticulate multiantigenic vaccines against H. 262–270. 63. A. Drug Deliv. A. Polym. Res. pylori: In vivo evaluation... Calvo. Intranasal immunization of mice against Streptococcus equi using positively charged nanoparticulate carrier systems. B. Control. Singh. Alonso.J. Brodin..E.Mar.. The enhancement of the immune response against S. M. Biomaterials 2009. 566–573... Coimbra.. 30.... Vila-Jato. 30. 66. Caetano. Gonçalves.A. Bouwstra. Polym. Nanotechnol. J. C.S. The manufacturing techniques of drug-loaded polymeric nanoparticles from preformed polymers.. Appl. M. Cadete. P. 2005. J. Almeida. [CrossRef] [PubMed] 34. L.. Jiskoot. [CrossRef] 35. A. 2012. Control. Sci. 10.. equi antigens through the intranasal administration of poly-epsilon-caprolactone-based nanoparticles.F. B.A.. 2001. 3. C. Sci. Release 2001. D.. Foged. B. Lacerda.. Almeida. H.. 110. Drugs 2016.

. Gupta. R. Ramanathan.E. B. H.. PLoS ONE 2008. S. Merwin. Kraan. D. Hennink.. 78. Sarmento. Sanchez. Tomar. N. A. Adv. De. Janes.. 82. 37.. N. Mishra. Ltd. Junginger. Pharm. McNeela. L.. [CrossRef] [PubMed] .Mar. Sci. 72.J. Polymer relationships during preparation of chitosan–alginate and poly-L-lysine–alginate nanospheres. M.. Tiwari. K. A. 90 28 of 30 36. Vaccine 2007. J.. Amidi.M. Verhoef. [CrossRef] 39. E. H. L.. Podda. B. Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: Enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan. 44.A. 21–32.. 1169–1173.. Kissel. M.R. A. Int. 392–400. Rappuoli. Rev. Effect of chitosan on epithelial permeability and structure. Tyagi. [CrossRef] 43. [CrossRef] [PubMed] 52. Evaluation of mucoadhesive PLGA microparticles for nasal immunization. 28.. Bouwstra... N. Van der Lubben.. 59–82. [CrossRef] [PubMed] 42. A.J.. Pharm..... Vaccine 2011. John Wiley & Sons. 12. 57.P. 62. Dixit. 14..... W.L. Azevedo. Lewis.. H.: Hoboken. 144–153. Junginger. Cadete. D. Del. M. Eur. Biopharm. J. A. W.. 43–48. Enhanced and enduring protection against tuberculosis by recombinant BCG-Ag85C and its association with modulation of cytokine profile in lung. Pawar.F. Chitosan-Based Systems for Biopharmaceuticals: Delivery. Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics..A. W.. 27. Pharm. S. A. M. Int. Development and characterization of chitosan coated poly-(ε-caprolactone) nanoparticulate system for effective immunization against influenza. 2004.S.. Jato. Drugs 2016. [CrossRef] [PubMed] 48. L.M. Goncalves. 3837–3844. J.H. 2001. Mastrobattista. M. AAPS J... Jiskoot... I. S. [CrossRef] 40. Rodrigues. 2002. Bernkop-Schnürch.. [CrossRef] [PubMed] 49. Sharma. and Polymer Therapeutics. 25. Alginate in drug delivery systems. K. Infect. I.. B. V. J. Yoo. M. Amorij.. Florindo. [CrossRef] [PubMed] 47.K. Eur. Jain..K. Control..D. Mes. Immunopharmacol.S.K. 2012. Kersten. Pharm. USA. V. J.. Hennink..J. R. 201–207... Targeting.C.E. Goyal. Release 2003. Ltd. Slütter. D. Development of a novel mucosal vaccine against strangles by supercritical enhanced atomization spray-drying of Streptococcus equi extracts and evaluation in a mouse model. Jain. 2012. M... Almeida. V. [CrossRef] [PubMed] 50.. Biopharm. B.. Behrens. W. Chitosan and derivatives for biopharmaceutical use: Mucoadhesve properties. Pizza. 2009. Chanput. Pharm. 22. B. Amidi.. 909–914. Neves. M. Junginger.P. [CrossRef] [PubMed] 55. E. Rao. J.C. G. Alonso.. Verhoef.J. NJ.. USA.G.. 101–112. 5341–5348. J. 2010. Jiskoot. J.. M. Ind. John Wiley & Sons. [CrossRef] 46.K.. Borchard.. L. Matos. [CrossRef] [PubMed] 38. V. 1999.. J. 159–180.. Mills. W. I. S.. S. 23.. 2014. Vaccine 2003. 2010. Chitosan and its derivatives in mucosal drug and vaccine delivery. 1–9. Figueiredo. Vila. 123–131.M. Vaccine 2012. 89. [CrossRef] [PubMed] 54. G. The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines. Dodane.. 29. 30.. N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: Biological properties and immunogenicity in a mouse model.A. Tafaghodia. Sarmento. A. [CrossRef] [PubMed] 51. M. Pharm.A. Rappuoli. 857–865. B. Tiago. Singh. Lehr.J. In Chitosan-Based Systems for Biopharmaceuticals: Delivery. Amin Khan. E. 1992.M. E. Cave. H. Illum. [CrossRef] [PubMed] 41. Vaccine 2004. C.M.D. Karlsen. Chitosan-based delivery systems for protein therapeutics and antigens.. P.D..V.H.. Dhar. R. NJ.. e3869. J. Jabbal-Gill. Baudner.. Gupta. Application of chitosan microspheres for nasal delivery of vaccines. H.. V. U. Giuliani. 2004.. Saluja..D. Wichers. Vyas. 182.. Schacht. J..G.K. Adv.. K. Int. Biotechnol. Drug Dev. Tønnesen. C. J.L. THP-1 cell line: An in vitro cell model for immune modulation approach. J. Cho. 3. Eur. 9026–9037.E. V. Drug Deliv. J.... 130–137. H..F. Leithner.. Mastrobattista.. Eisenach. Padrela.E. V.A. H. A. Robinson. T. In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers.C. Vaidya. 14. Eds. W. Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. H. H. J. [CrossRef] [PubMed] 53. 21. 621–630. Theus. E. Katoch. K. Activated THP-1 cells: An attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates. 2012. J. Mangal. R. Dey. G.: Hoboken. [CrossRef] 45.. A. pp. Kang. Targeting and Polymer Therapeutics. Jiskoot. Immun.

interactions with food components and applicability as a coating on fruit and vegetables.. F. Veiga. J. Methods for nano-particle based vaccine formulation and evaluation of their immunogenicity. G. 2198–2206.B. 2006. 40.M.E. Rev. Díaz.K.... 703–714.L.. J. Rafiei. Stringer.M. Johansen. [CrossRef] 78. [PubMed] 75. M. Dobakhti. Mottram. V. Bleck. E. J.. H. L. Pharm. P. Pharmacol.. Eds. S. E.. Fretz. 305–320. S. 23. Microencapsul. D. B.M... [CrossRef] [PubMed] 62. Dehpour. Release 2004.. Kersten. Verhoef. Carbohydr. 68. 11..Y. Microbios 1988. 77.. Release 2010. 24.A. 2005. 460–466. Bond.. 15. Groves.M.. S. Immunity in response to particulate antigen-delivery systems. Chitosan: Antimicrobial activity. J. Microencapsul. Drugs 2016. [CrossRef] 67. Technology. 2003. Cell Biol. 527–530. Rev.. K. Griffiths. P.. G. S. 81–92. Tice Substrain. Springer-Verlag: New York. Bramwell. Tice substrain... Pharm. A. 2004. Int. Dodov. Res.. E. 88. Ferreira.C. Méndez-Vilas. Pietersz. USA. FORMATEX: Badajoz.. G. [CrossRef] 71.G.. V.-H. Zhang. Rev. Takka. [CrossRef] 70. Van der Lubben. 34. I. 8.. Kovalainen. [CrossRef] [PubMed] 76. 141. F. N. Study on antimicrobial activity of chitosan with different molecular weights.R. Groves. Particulate delivery systems for vaccines: What can we expect? J. J.. 265–276. Senti. Effect of isotonic solutions and peptide adsorption on zeta potential of porous silicon nanoparticle drug delivery formulations.. Methods 2006. Anes.. J. 2003. Lansing. Wu. Desjardins. J. Klegerman. M. Bettencourt. Riikonen. J. G. M. G. J.. 285–300. AAPS PharmSciTech 2010. Järvinen.. J. Microparticles and polymers for the mucosal delivery of vaccines. Control. M. 2010. Cell. Crimeen-Irwin.. Methods 1983. 827–834. Plebanski. Adv.. 54. 57.. Neufeld. Xiang. J. M.. Drug Deliv. Factorial design analysis and optimisation of alginate-Ca-chitosan microspheres. Jordao. Sarmento. Microbiol. 2012. M. Nanosystem drug targeting: Facing up to complex realities. Ajdary. M. Kalkanidis. Spain. F. 2013.. D. Curr. 529–548. M. 614–621. Res. 2010. Polyelectrolye complex formation between alginate and Chitosan as a function of pH.E. Cook.. 20–29. The surface charge of cells of Mycobacterium bovis BCG vaccine.A. M.J.. Storni. Microscopy: Science... S. L. Bell.F. Goracinova.. Yu.M.... F. A. In Bergey’s Manual of Systematic Bacteriology. L. Taxonomic outline of the prokaryotes. Ardipradja. Alginate/chitosan nanoparticles are effective for oral insulin delivery. 230–236. 717–728. [CrossRef] [PubMed] 73. 844–854. P. Phagocytosis: Latex leads the way.. Lehto.. Kaasalainen. Salonen. J. G. 65. 96. On the killing of mycobacteria by macrophages. J.J. [CrossRef] . Polym. Nanobead-based interventions for the treatment and prevention of tuberculosis. Simsek-Ege.K. Nyström. [CrossRef] 66. Ribeiro. Adv. Immunol.Mar.. Stabilizing effects of calcium alginate microspheres on Mycobacterium bovis BCG intended for oral vaccination. Zhu. T. Mosmann. B. Perrie. Pharm. 21. K. Garrity.G..E. [CrossRef] [PubMed] 69. B. F. Geskovski.S.. A. 5. J. Lin. 30. 346–351. 90 29 of 30 56. [CrossRef] [PubMed] 58. L. Sampaio.. Sung.. 333–355. Pires. 607–610. Khuller... Beuvery. NY. A novel pH-sensitive hydrogel composed of N. P.. 498–503. G. A. G. Zhang. Crcarevska. 191–195.. 2008. 1998. 2003. Hagan.. Florence. Vaccine 2003.O. [CrossRef] [PubMed] 65. A. Makraduli. Chen. 1988. Pharm. A. Vermeulen. Mäkilä.. [CrossRef] [PubMed] 64. A. 2004..A. Y. Y. 10. Anes. S. Food Microbiol.. Junginger. Size characterization of Mycobacterium bovic BCG (Bacillus Calmette Guérin) Vaccine. G.O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. Ruenraroengsak. [CrossRef] [PubMed] 63. D. Gürel. K.. Carmo.. Control. Polym. [CrossRef] [PubMed] 59. Drug Deliv. 1400–1408. pp.. [CrossRef] 74. C. M. E. Rahimi. Griffiths. J. Nat. Chitosan microparticles for mucosal vaccination against diphtheria: Oral and nasal efficacy studies in mice. Mi. M..T. Kündig. Sci. Applications and Education. Debevere.T. [CrossRef] [PubMed] 57. J. N.W. T... 2007. T. M. Y. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. H.. Appl. Microbiol. Opin. T. R. Sable. P.. 58. Griffiths. Herzig.. Zheng. Evaluation of chitosan/alginate beads using experimental design: Formulation and in vitro characterization. Devlieghere. Lilburn. A. K.D. [CrossRef] [PubMed] 60. [CrossRef] [PubMed] 61. 2006. 53. 431..M.. [CrossRef] [PubMed] 72. F. L. 21. 14. C. 55–63..-P. Taghikhani. Mayorga..

Polím. C. Assis. [CrossRef] [PubMed] 81.. M. 64.G.. Seagar. A.B. 778–779. 55. Liu. Liu. De Britto. Thermochim. B. Thermogravimetric and FTIR studies of chitosan blends. K. Park. X. Biotechnol.C.org/licenses/by/4. D. Pathol. Effect of MW and concentration of chitosan on antibacterial activity of Escherichia coli. C. Watt. Goy. Doig.-G.. 2009. 153–166. Tecnol.J. X. [CrossRef] 80. Polym. Basel.. 2009. 2.. Pawlak. A review of the antimicrobial activity of chitosan. [CrossRef] 82.. Acta 2003. 19. Mucha. [CrossRef] [PubMed] 83. View publication stats . L. Meng. R. D.. Sahl. J.0/). Chen. Yu. 2002. Microb. Chitosan and its antimicrobial potential—A critical literature survey. Switzerland. 396.. Mar. H. 186–201. Liu. H.. A.. N. 14. 2006. 90 30 of 30 79. Raafat. C. Drugs 2016. licensee MDPI. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons. Ciênc. O. 60–65. Clin.. Forbes.. The efficacy of the heat killing of Mycobacterium tuberculosis. Carbohydr. [CrossRef] © 2016 by the authors. 241–247..L.