Bacteria microencapsulation in PLGA microdevices by supercritical emulsion extraction

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Bacteria microencapsulation in PLGA microdevices by supercritical emulsion extraction G. Della Porta a,b,∗ , F. Castaldo a , M. Scognamiglio a , L. Paciello a , P. Parascandola a , E. Reverchon a a b

Dipartimento di Ingegneria Industriale, Università di Salerno, Via Ponte don Melillo, Fisciano (SA), Italy Laboratorio di Ingegneria Cellulare e Molecolare, DEIS (sede di Cesena), Università di Bologna, Via Venezia 52, Cesena (FC), Italy

a r t i c l e

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Article history: Received 13 October 2011 Received in revised form 19 December 2011 Accepted 20 December 2011 Keywords: Supercritical fluid Emulsion Microdevices PLGA Bacteria encapsulation

a b s t r a c t Cell microencapsulation continues to hold significant promise for biotechnology and medicine and is considered an important tool for tissue engineering. Encapsulated cells would also provide a source of sustained continuous release of therapeutic products for longer durations at the site of implantation. The present work investigates the possibility of prokaryotic cells microencapsulation by Supercritical Emulsion Extraction (SEE) technology; Lactobacillus acidophilus was selected as a model bacterium and poly-lactic-co-glycolic acid (PLGA, 75:25) was chosen as biopolymer because FDA approved in devices for biomedical applications. A double emulsion (w1 -o-w2 ratio 2:18:80) was used with an internal water phase (w1 ) composition of L. acidophilus suspended in MRS broth plus the 0.4% of poly-vinyl alcohol (PVA), as surfactant; the best overall cell mass content was found to be not higher than 10 mg/mL (that correspond to 7.5 × 106 UFC/mL). Other emulsion phases were: o-phase containing ethyl acetate (EA) and PLGA at 10% (w/w) and w2 -phase of water plus 0.6% of PVA (w/w). This emulsion treated by SEE at 90 bar and 37 ◦ C for 30 min allowed the formation of PLGA microcapsules with a mean size of 20 ␮m (±10 ␮m) loaded with the 0.6% (w/w) of microorganism with an excellent encapsulation efficiency (80%). Size and morphology of the produced microdevices were monitored by laser scattering and by SEMEDX analyses and confirmed SEE as an innovative and efficient encapsulation technology. Particularly, the microspheres were constituted by a PLGA wall containing the cells entrapped into the polymeric matrix. Cell viability less than 5% (w/w) with respect to the loaded microorganism was also evaluated; nevertheless the biodegradable microdevice produced may be particularly interesting for several biotechnological application in which mainly the killed vectors is used as bioactive signal delivery. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Cell microencapsulation in biodegradable devices is considered an important challenge for tissue engineering and regenerative medicine [1]. Indeed, cells targeting problems (live or death) can be overcame by their delivering using biopolymer microcapsules which can protect the encapsulated materials from the harsh external environments [2,3] or address it to a more specific target [4–6]. Microspheres loaded with cells have been also proposed as activate biopolymer scaffolds for tissue engineering applications [7,8] or as immunoprotected implants for cell-based therapy [9]. Encapsulated cells may also provide a source of sustained continuous release of therapeutic products for longer durations at the site of implantation [10]. For example, bacteria can be genetically

∗ Corresponding author at: Dept. of Industrial Engineering, University of Salerno, Via Ponte Don Melillo, 84084 Fisciano (Sa), Italy. Tel.: +39 089 964104; fax: +39 089 964057; mobile: +39 320 7979003. E-mail address: [email protected] (G. Della Porta). 0896-8446/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.supflu.2011.12.020

engineered to synthesize products with high therapeutic potential and used as a potential delivery vector for various antigens or therapeutic and immunomodulatory proteins [11]. In vitro or with animal models studies demonstrated that Lactococcus lactis has emerged as potential delivery vector of different antigens and, in some specific cases, also the killed bacterium can be an interesting vector capable to produce comparable responses to those elicited by live bacteria [12]. Although native L. lactis is a generally recognized as safe (GRAS), the genetically modified versions have to be assessed for safety and biological containment. From this perspective, the use of dead L. lactis vectors seems to be a safer option if the efficacy is retained, because there is no risk of spreading recombinant DNA in the environment. Several technologies have been tested for the production of bacteria loaded microdevices, such as spray-drying, extrusion, phase separation and solvent extraction/evaporation of emulsions. Almost all of these technologies were tested on probiotics bacteria mainly to develop probiotic health based product. However, none of these reported methods has resulted in a large number of shelf-stable cells necessary for use in industry [13]. Mainly

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problems of low encapsulation efficiency and reduction of cell viability were reported [14,15]. For example, the use of spray drying for Lactobacilli and Bifidobacteria microencapsulation was reported for a number of different strains [16,17] including Lactobacillus acidophilus [18]; however, most of the bacteria do not survive at temperature or osmotic extremes to which they are exposed during the spray drying or emulsification process and, in several cases, reduced encapsulation efficiency was also reported. A different cell coating technique was proposed by Picot and Lacroix [19,20] using an emulsion containing milk fat droplets and whey protein polymers with freeze-dried bacteria, then spray dried. The direct dispersion of fresh cells in a heat-treated whey protein suspension followed by spray drying was suggested as the less destructive microencapsulation method, with survival rates of 26% for Bifidobacter breve and 1.4% for the more heat-sensitive Bifidobacter longum [21]. Several biodegradable polymer matrixes have been also tested for bacteria encapsulation such as: alginate systems [22], cellulose acetate phthalate [23], proteins and polysaccharide mixtures [24], chitosan [25]. Poly-lactic-co-glycolic acid (PLGA) was never used until now because almost all biopolymer matrix reported above were selected for food industry applications; whereas, the encapsulation into PLGA microdevices may be much more interesting for biotechnological and biomedical applications [26]. The most common method of PLGA microdevices preparation is the solvent evaporation/extraction of emulsions; this technology may require elevate temperatures or reduced pressures and long processing times (several hours), that may produce particles aggregation, as well as, cells damage [27]. Supercritical fluid technologies were also proposed to produce PLGA microdevices. Particularly, RESS technology showed problems of low solubility of almost all PLGA co-polymers in SC-CO2 that will prevent affordable process yields [28]; whereas, the even low solubility of SC-CO2 in the PLGA is again the main problem when using the SAS technology because it causes the precipitation of large polymer aggregates [29]. More recently, Bifidobacteria encapsulation into PVA/PVA complex matrix by supercritical fluid was also reported followed by a milling of the obtained complex [30]. Supercritical Emulsion Extraction (SEE) technology was recently proposed for the production of biopolymer microspheres by several authors from oil-in-water emulsions [31,32]. The authors reported a better control of microspheres sizes and distributions, as well as, excellent drug loading achieved by SEE with respect to the conventional extraction/evaporation technology; the innovative process was also characterized by mild temperature conditions. The very good process performances were justified considering supercritical fluid properties such as lower viscosity and higher diffusivity that will improve the mass transfer and reduce the processing times during the extraction of the oily dispersed phase [33,34]. The present work aims to investigate the possible use of SEE technology, as a low impact method, for bacterial cells encapsulation into PLGA microdevices intended for biomedical and pharmaceutical use. L. acidophilus was selected as a model bacterium because of its high sensitivity to temperature or osmotic variations. Our aim was also to explore SEE process conditions that may allow high encapsulation efficiency of intact bacteria even with low cell viability, considering that genetically modified bacteria may eventually be also used as antigen vectors, also when dead. In this sense, double water–oil–water emulsions with different internal water phase compositions and biopolymer concentrations in the oily phase were tested to monitor the effects of these parameters on droplets stability, microspheres morphology and cells loading. The SEE process parameters like operating pressure and temperature, flow rate and contacting time between the emulsion and supercritical carbon dioxide (SC-CO2 ) were also studied with respect to microsphere size distribution and to cells

encapsulation efficiency. A comparative study between the characteristics of the microspheres obtained by SEE and those produced by conventional solvent evaporation (SE) was also proposed. Cells dispersion and viability into microspheres were also monitored. 2. Experimental methods 2.1. Materials CO2 (99.9% SON Naples, Italy), polyvinyl alcohol (PVA, mol. wt.: 30,000–55,000, Aldrich Chemical Co.), ethyl acetate (EA, purity 99.9%, Aldrich Chemical Co.), poly (lactic/glycolic) acid (PLGA, 85:15 mol. wt.; 20,000–60,000 Aldrich Chemical Co.) were used as received. L. acidophilus ATCC 43121 was grown at 37 ◦ C for 24 h in Erlenmayer flasks containing 200 ml MRS Broth (BD 288130), using aerobic conditions. 2.2. Biomass production To obtain viable cells of L. acidophilus, two-step cell propagation was set up in 500 mL Erlenmeyer flasks, incubated at 37 ◦ C and 150 rpm in an Orbital Incubator (Stuart Scientific S150). In the first step, 1 mL of a frozen culture (stored at −80 ◦ C in 20.0%, v/v glycerol) was propagated in 200 mL MRS broth. During exponential growth, an aliquot of broth culture was withdrawn to be employed as inoculum in the second step propagation flasks. In the latter case, L. acidophilus cells were allowed to grow for 24 h in 200 mL MRS broth to achieve a viable biomass suspension having an optical density of 1.8 at 590 nm. Beyond this OD value, cell conglomeration phenomena begin to occur, which reduce cell viability. The obtained biomass was then collected by centrifugation at 6500 rpm for 20 min and re-suspended in fresh MRS broth to be processed and entrapped in PLGA matrix. 2.3. Emulsions preparation Different double emulsions were prepared to optimize the composition of each phase: 5 or 10 mg/mL of live cells were presuspended into MRS broth/PVA solution or aqueous PVA solution; a fixed amount of these suspensions was then added into EA/PLGA solutions and sonicated for 2 min (Digital Sonifer Branson mod. 450). This primary emulsion was added into a known amount of aqueous PVA solution to form the secondary emulsion using a highspeed stirrer (mod. L4RT Silverson Machines Ltd., United Kingdom) operating at 800 rpm for 3 min at 10 ◦ C, controlled using an ice bath. 2.4. Microspheres preparation by conventional solvent evaporation EA was evaporated from the emulsions for 60 min at 30 ◦ C under controlled and mild vacuum (170 mmHg, rotating evaporator) under moderate stirring. During the evaporation, the emulsions were swept by a continuous nitrogen flow at constant flow rate (70 L/h). 2.5. SEE apparatus: description and procedure In a typical experiment, 40 g of emulsion were placed into the 0.25 dm3 cylindrical stainless steel vessel. SC-CO2 was delivered using a high pressure diaphragm pump (Milton Roy, model Milroyal B, Point Saint Pierre, France) and was bubbled into the extraction vessel at a constant flow rate (0.1–0.5 kg/h), through a cylindrical stainless steel dispenser located at the bottom of the extractor. The cylindrical dispenser maximizes the contact between the two phases during the extraction. Temperature was maintained constant using an air-heated thermostated oven. A separator located

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syringe, which was then immersed for 2–3 min in liquid nitrogen to solidify the gelatine suspension. The solid gelatine was then precut in samples of 5 mm long. These samples were then mounted on a refrigerated stub using a small amount of Neg 50, immersed in the pre-chilled iso-pentane and sectioned in a cryostat (mod. HM 550, MICROM International Gmbh) at 40 ◦ C, using a pre-chilled blade. The sections were cut and prevented from curling during sectioning using a small pre-chilled brush. Sections with a thickness of 20 ␮m were used for the microscopic measurements. 2.8. Cell dispersion in the microspheres Cells dispersion in the microspheres was evaluated using an Energy Dispersive X-Ray analyzer (EDX mod. INCA Energy 350, Oxford Instruments, Witney, UK), using the signal of Phosphorus that is present only in the cell. Before the evaluation of the elemental composition, the samples were coated with chromium (layer thickness 150 A) using a turbo sputter coater (mod. K575X, EmiTech Ashford, Kent, UK). Fig. 1. Description of the apparatus: CO2 cylinder, HP, high pressure diaphragm pump, SEE, high pressure vessel, S, separator for oily phase recovery.

downstream the micrometering valve was used to recover the liquid solvent extracted and the pressure in the separator was regulated by a backpressure valve. At the exit of the separator a rotameter and a dry test meter were used to measure the CO2 flow rate and the total amount of CO2 delivered, respectively. When the extraction process was complete, the suspension was recovered from the extractor vessel for further processing. Particles were washed several times by centrifugation with distilled water and collected by membrane filtration. Particles were also dried for morphological studies or incubated in MRS broth for viability evaluations. A schematic representation of the proposed process and the apparatus used is reported in Fig. 1. 2.6. Droplets and microspheres morphology and size distributions The microorganisms and the droplets formed in the emulsions were observed using an optical microscope (mod. BX 50 Olympus, Tokyo, Japan) equipped with a phase contrast condenser. Field Emission-Scanning Electron Microscope (FE-SEM mod. LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany) was used to study the morphology of the collected microspheres. Dried powder was dispersed on a carbon tab previously stuck to an aluminum stub. ˚ Samples were coated with gold–palladium (layer thickness 250 A) using a sputter coater (mod. 108 A, Agar Scientific, Stansted, UK). Droplet size distributions (DSD) and particle size distributions (PSD) were measured by dynamic light scattering (mod. Mastersizer S, Malvern Instruments Ltd., Worcherstershire, UK). The Mastersizer S software uses Mie theory to produce an optimal analysis of the light energy distribution and to obtain the size distribution of the particles. Analyses were performed just after the preparation of emulsions and microsphere suspensions using several milligrams of each sample and repeated ten times. 2.7. Microspheres sectioning The microspheres sectioning procedure was adapted from Ehtezazi et al. [35]. Microspheres were dispersed for 2–3 h in an aqueous solution containing gelatine (20%, w/v control ref. 30) and glycerine (5%), to allow the complete occupation of the microspheres internal microporosity by the aqueous medium, this occupation was necessary to avoid internal pores distortion during the sectioning. The suspension was then drawn into a 2.5 mL

2.9. Cell viability evaluation PLGA microcapsules were dispersed in a MRS broth for 36 h at 37 ◦ C. Then, aliquots (50 ␮L) of these cell suspensions were spread on YPD or MRS agar plates and incubated in the aerated Heraeus incubator at 37 ◦ C for 48 h to evaluate the Colony Forming Units (CFU) originated from the viable cells. 3. Results and discussion 3.1. SEE process parameters selection SEE operating pressure and temperature conditions were selected to enhance the extraction of the oily dispersed phase of the emulsion; whereas, the SC-CO2 flow rate was chosen to avoid emulsion/suspension loss by washing out in the SC-CO2 stream. In a previous work [31] it was reported that EA/CO2 mixture critical pressure at 37 ◦ C is of 80 bar with a CO2 molar fraction of 0.9; at these operating conditions water is only slightly soluble in SC-CO2 ; whereas, EA is fully miscible. As a consequence, operating conditions explored in this work were 90 bar (slightly higher than the mixture critical point) and 37 ◦ C to prevent microorganisms suffering by its warming up. It has also to be considered that the compressed CO2 may have a “bactericidal effect”, as reported by several authors [36,37]. However, it was also indicated that it is not the pressurized carbon dioxide to be really active in enzymes denaturation or bacteria inactivation but the fast depressurization step at the end of the process. Moreover, the effective sterilization with compressed CO2 was also obtained by combining a thermal effect or adding others agents, such as peracetic acid. Nevertheless, to avoid any adverse effect in this sense, the SEE apparatus was designed to allow very slow compression/decompression steps of the suspension. At the operating pressure and temperature conditions reported above, SC-CO2 is expected to be an excellent solvent for EA and, after contact with emulsion, to extract the organic solvent, producing the polymer hardening into microspheres. A SC-CO2 flow rate of 0.4 kg/h was used for 30 min for treating 40 g of emulsion. Operating at these conditions, the ethyl acetate residue into the recovered suspension was less than 50 ppm. Higher flow rates of 0.6 and 0.8 kg/h were also tested and considered not suitable, due to a large entrainment of emulsion in the gaseous stream. Shorter processing times of 10 and 20 min were also investigated, but, in both cases ethyl acetate elimination was less efficient and solvent residues of 354 ppm and 825 ppm, respectively, were measured.

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Fig. 2. (a) OM image of w1 -o-w2 emulsion with ratio of 4:16:80 (microorganism concentration in w1 of 5 mg/mL). (b) SEM image of PLGA microspheres after SEE process operating at 90 bar and 37 ◦ C, with a SC-CO2 flow rate of 0.4 kg/h for 20 min.

Fig. 3. (a) OM image of w1 -o-w2 emulsion with ratio of 2:18:80 (microorganism concentration in w1 of 5 mg/mL). (b) SEM image of PLGA microspheres after SEE process operating at 90 bar and 37 ◦ C, with a SC-CO2 flow rate of 0.4 kg/h for 20 min.

3.2. Emulsions optimization for L. acidophilus encapsulation The droplets in the emulsion were produced in the range of 10–40 ␮m to be large enough to encapsulate bacterial cells that are of about 1–2 ␮m in size. Different emulsion compositions were tested to find the optimal phase ratios and the optimal concentration of each component. In some preliminary experiments, distilled water with a PVA concentration of 0.4% was used as the water internal phase, but this emulsion failed in entrapping bacteria inside the droplets. In addition, monitoring the cell viability, it was observed that cell survival in distilled water was much reduced (vitality decreased to 50% in 24 h). In the subsequent experiments, a MRS broth was used as the internal water phase, with the same PVA concentration, as surfactant. The cell mass content charged in the internal water phase was reduced to 5 mg/mL (that correspond to 3.7 × 106 CFU/mL). The oily phase contained 10% (w/w) of PLGA in EA and the external water phase was formed by distilled and sterilized water with the 0.8% of PVA, as surfactant. Emulsions with different internal water phase ratios, namely of 4:16:80 and 2:18:20 (w1 -o-w2 ) were also compared. When the 4:16:80 ratio was used, the emulsion was unstable as is also illustrated in the OM image reported in Fig. 2a, in which it is well visible the migration of the internal water phase to the external one. As a consequence, open microcapsules were produced after the SEE process and almost all bacteria were recovered outside the particles. An example of the produced material is illustrated in the SEM image reported in Fig. 2b. When the w1 -o-w2 ratio of 2:16:80 was tested, stable droplets were produced, as illustrated in the OM image reported in Fig. 3a, in which spherical droplets

that contain cells inside are evident. Drying this emulsion by SEE, spherical microcapsules were obtained, as illustrated in the SEM image reported in Fig. 3b. An emulsion with a w1 -o-w2 ratio of 2:16:80 was also tested, increasing the L. acidophilus concentration in the internal water phase to 10 mg/mL (that correspond to 7.5 × 106 CFU/mL); in this case, probably due to the higher cells concentration, the bacteria were extruded from the droplets and confined on the microspheres surface, as can be observed in Fig. 4 where a SEM image of the PLGA microparticles obtained is illustrated. All the emulsions described above were processed also by conventional solvent evaporation (SE) and for all the emulsion composition explored the microspheres obtained by SE were characterized by an open and irregular morphology, with a large amount of cells outside the particles. Particularly, when the optimized emulsion composition of w1 -o-w2 ratio 2:16:80 (with cell content in w1 of 5 mg/mL) was treated by SE, mainly empty and concave particles were produced. An example of these concave and collapsed microparticles is illustrated in the SEM image reported in Fig. 5. The reason of the observed morphologies was well described by Rosca et al. [38] and it was mainly due to the low stability of the double emulsion during the SE treatment. Indeed, the authors reported that when double emulsions are processed by conventional solvent evaporation, since the process takes several hour, the loss of the water internal phase can easily occur, generating concave and collapsed shaped particles that are also expected to be empty.

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30 25 20 15 10 5 Fig. 4. SEM image of PLGA microspheres obtained operating at 90 bar and 37 ◦ C, with a SC-CO2 flow rate of 0.4 kg/h for 20 min. They were produced using an emulsion with w1 -o-w2 ratio of 2:18:80 and with a microorganism concentration in the internal w1 phase of 10 mg/mL.

DSD and PSD curves, obtained by laser scattering analysis for each emulsion and microparticle suspension studied were also used, as an indirect method, for encapsulation efficiency evaluation. Indeed, a simple bacteria suspension showed a Mean Diameter (MS) of 1.2 ␮m with a Standard Deviation (SD) of ±0.5 ␮m, whereas, oily droplets and derived PLGA microspheres where produced with mean sizes between 20 and 50 ␮m; therefore, when bacteria encapsulation was not good, bimodal curves were expected. Examples of particle size distributions obtained are illustrated in Fig. 6a and b. Particularly, in Fig. 6a is reported the histogram representing the PSD of the microspheres obtained from the not stable emulsion with w1 -o-w2 ratio of 4:16:80 (microorganism concentration in w1 of 5 mg/mL) also illustrated in Fig. 2a and, in this case, the presence of a bacteria suspension not well entrapped into the microspheres can be easily recognized by the typical bimodal evolution of the diameter populations. In Fig. 6b is reported the histogram representing the PSD of the microspheres obtained from the stable emulsion with w1 -o-w2 ratio of 2:18:80 (microorganism concentration in w1 of 5 mg/mL) also illustrated in Fig. 3a and, in this case, only one peak is present confirming the absence of the free bacteria in suspension. The microspheres produced in this last case showed a MD of 20 ␮m and SD of ±10 ␮m. This emulsion

Fig. 5. SEM image of PLGA microspheres obtained by the conventional solvent evaporation process.

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Fig. 6. (a) PSD of the microspheres obtained from not stable emulsion (w1 -o-w2 ratio: 4:16:80 and cell concentration of 5 mg/mL); (b) PSD of the microspheres obtained from stable emulsion (w1 -o-w2 ratio: 2:18:80 and cell concentration of w1 of 5 mg/mL).

composition was confirmed to be the best for bacteria entrapment after SEE processing. To further evaluate the bacteria spatial distribution inside the particles, the elemental composition of microspheres was studied by Energy Dispersive X-Ray (EDX) analyzer integrated in the SEM apparatus. Cells contain in a relatively high amount the Phosphorus (P) that is not present in PLGA; therefore, it can be used to indicate the location of the microorganism into the microdevices. In Fig. 7 SEM image of the microspheres with elemental maps of some constitutive elements (carbonium, oxygen and phosphorous) are reported. It is evident that phosphorus (mapped in red) is uniformly spread over all the microspheres; i.e., cells should be uniformly distributed into the microspheres. The EDX relative amount of the phosphorus with respect to the other elements indicates the presence of almost 0.8% of phosphorus with respect to the overall carbon atoms identified in the sample confirming the efficiency of cell loading into microspheres. Microcapsule internal structure was also investigated by sectioning them using a stereological method; then, selected sections

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Fig. 7. EDX image of PLGA microspheres charged with 0.6% (w/w) of cells obtained operating at 90 bar and 37 ◦ C, with a SC-CO2 flow rate of 0.4 kg/h for 20 min. Phosphorus was highlighted in red, carbon in green and oxygen in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

were observed by SEM. An example of the SEM image of the section obtained is illustrated in Fig. 8; in the figure it is possible to observe the bacteria cells inside the microcapsule. Bacteria viability was also monitored for these microcapsules by their incubation into a MRS broth for 36 h; it resulted to be less than 5% (w/w) with respect to the loaded microorganism.

4. Conclusions and perspectives The production of spherical microdevices formed by PLGA, containing the bacteria entrapped into the polymeric matrix was demonstrated to be a possible application of the SEE technology processing a double water–oil–water emulsion with supercritical carbon dioxide at 90 bar and 37 ◦ C for 30 min. A particle size target of 20 ␮m as mean size with an optimum percentage of 0.6% (w/w) cell loaded were successfully obtained (encapsulation efficiency 80%). Nevertheless very low cell viability was monitored suggesting that the described technology can be applied, for example, in the encapsulation of genetically modified bacteria especially when dead cells must be used in order to retain almost all the therapeutically efficacy as biological signals and to avoid any others adverse effects. However, future perspectives of this work can be the improvement of bacteria survival through the addition of protectants to the liquid media in which they are retained in emulsion prior to SEE treatment and/or growth promoting factors including various probiotic/prebiotic combinations. References

Fig. 8. SEM images of PLGA microcapsule section obtained using SEE technology.

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