Controlling the Growth of Staphylococcus epidermidis by Layer-By

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Biological and Medical Applications of Materials and Interfaces

Controlling the Growth of Staphylococcus epidermidis by Layer-by-Layer Encapsulation Alain M. Jonas, Karine Glinel, Adam Behrens, Aaron C Anselmo, Robert Langer, and Ana Jaklenec ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01988 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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ACS Applied Materials & Interfaces

Controlling the Growth of Staphylococcus epidermidis by Layer-by-Layer Encapsulation Alain M. Jonas,*,1,2 Karine Glinel,*,1,2 Adam Behrens,2 Aaron C. Anselmo,2,3 Robert S. Langer,2 Ana Jaklenec2

1

Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Croix du Sud 1/L7.04.02, Louvain-la-Neuve, 1348, Belgium 2

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, USA 3

Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Abstract Commensal skin bacteria such as Staphylococcus epidermidis are currently being considered as possible components in skin-care and -health products. However, considering the potentially adverse effects of commensal skin bacteria if left free to proliferate, it is crucial to develop methodologies that are capable of maintaining bacteria viability while controlling their proliferation. Here, we encapsulate S. epidermidis in shells of increasing thickness using layer-by-layer assembly, with either a pair of synthetic polyelectrolytes or a pair of oppositely-charged polysaccharides. We study the viability of the cells and their delay of growth depending on the composition of the shell, its thickness, the charge of the last deposited layer, and the degree of aggregation of the bacteria which is varied using different coating procedures – among which is a new scalable process that easily leads to large amounts of non-aggregated bacteria. We demonstrate that the growth of bacteria is not controlled by the mechanical properties of the shell but by the bacteriostatic effect of the polyelectrolyte complex, which depends on shell thickness and charge of its outmost layer, and involves the

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diffusion of unpaired amine sites through the shell. The lag times of growth are sufficient to prevent proliferation for daily topical applications.

Keywords Coated microorganism; Layer-by-layer assembly; Commensal skin bacteria; S. epidermidis; polyelectrolytes; chitosan.


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1. Introduction The importance of microbiota in the regulation of human health has been proposed more than a century ago by Döderlein.1 The microbiota protects its host by controlling the population of pathogenic microorganisms through the secretion of antimicrobial molecules, by sheer niche occupation, or by specifically stimulating the immune system of its host.2 The microbiota also plays key roles in enabling and facilitating host biological functions such as digestion and metabolism. There is currently increasing suspicion that alterations of the microbiota by changes in diet and hygiene, or by an indiscriminate use of antibiotics, may contribute to the development of auto-immune pathologies, allergies and inflammatory disorders.2 The delivery of probiotics to the gastrointestinal tract is a typical example of a strategy used to restore healthy populations of dysbiotic microbiota.3-6 In addition to the gastrointestinal tract, the urogenital and respiratory tracts, the mouth and nasal cavities, and the skin also host specific microbiotae.7 Being the largest organ of the human body and in direct contact with the environment, the skin is an especially important ecological niche for a wide variety of microorganisms. The beneficial effect of some skin commensal bacteria has been highlighted in recent years; for instance, Staphylococcus epidermidis, a major Gram-positive bacterium of the human skin, has been shown to deter nasal colonization by Staphylococcus aureus,8 to inhibit inflammation induced by Propionibacterium acnes (the main cause of inflammation in acne vulgaris),9 to secrete selective antimicrobial agents, including acidic substances that decrease skin pH and molecules inhibiting quorum sensing, 10 to stimulate the secretion of antibacterial peptides by keratocytes,10 and even to counteract the influenza virus.11 S. epidermidis has also been shown to modulate the immune response of its hosts.12,13 Nevertheless, for all their potential benefits, commensal skin bacteria may also pose p. 3 ACS Paragon Plus Environment


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serious threats,14 for instance when they pass the epidermal barrier and penetrate into the blood stream, which can lead to sepsis. This is certainly the case for S. epidermidis, which has been described as living at the edge between commensalism and pathogenicity,15 acting as guardian or pathogen depending on circumstances,13 and as such, has been defined as an "accidental pathogen".16 Skin dysbiosis, i.e., the perturbation of the skin microbiota, may be related to pathologies such as psoriasis, acne, or atopic dermatitis (eczema).9,12,17 Therefore, it was concluded by Grice et al.18 that maintaining an healthy skin requires not only therapies for the inhibition of the growth of pathogenic bacteria, but also for the promotion of symbiotic bacteria. As a result, cosmetic formulators are currently testing skin-care products containing live bacteria.19 However, for the previously-mentioned reasons, this should only be done while mitigating the potentially negative effects of commensal skin bacteria if left free to proliferate. In this context, it becomes timely to develop methodologies capable of maintaining commensal skin bacteria activity while regulating their proliferation. Some bacteria have the capability to enter a "viable but nonculturable" (VBNC) state, from which they can be resuscitated upon proper stimulation.20 However, bacteria in a VBNC state face significant challenges in detection and characterization,21 and their inclusion in therapeutics, supplements, or cosmetics will face challenges in regulation and product consistency. As such, other, more universal, methods are needed to ensure proper quality control and characterization when including live microbes in products designed to be interfaced with skin. One such method is the encapsulation of bacteria in a porous shell through which nutrients and products can diffuse, while still maintaining viability but delaying bacterial growth. Methods of cell encapsulation have been reviewed;22-31 they have so far mainly concentrated on yeast cells, although bacteria such as (spores of) p. 4 ACS Paragon Plus Environment

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Bacillus subtilis,32-34 Allochromatium vinosum,35 Lactobacillus acidophilus,6 Escherichia coli,36-40 Alcaligenes faecalis,41, cyanobacteria,42 Micrococcus luteus,43 and Bacillus coagulans3 were investigated as well. Shells of different chemical composition have been proposed, ranging from hard materials (silica,44-47 calcium carbonate,39,48 calcium phosphate,41,49 gold,50 metal-organic frameworks43) to softer organic layers based on layer-by-layer (LbL) assemblies,3,6,32,33,37,38,51-56 polydopamine,57 or tannic acid.58,59 A relatively large experimental body has been collected for yeast on the delay of germination upon encapsulation. Thick inorganic shells of ca. 1 µm thickness lead to the complete blocking of germination until the shell is dissolved.49 In contrast, thin organic coatings result in a delay of germination which increases with shell thickness, with only a limited dependence on shell composition.54-58. For instance, a 80 nm-thick polydopamine coating delays germination by 84 h,57 a 40 nm-thick coating of crosslinked (polyethylene imine/hyaluronic acid) retards germination by 36 h,56 and a 14 nm-thick hydrogen-bonded LbL by 22 h.58. Surprisingly, thin silica layers appear to be much less efficient in retarding the germination of yeast, with only 2.5 h delay for a shell of ca. 30 nm thickness.45 In contrast, much less data is available for bacteria: a (glycol chitosan/alginate)3 LbL-assembled organic shell of ca. 7 nm dry thickness was shown by Anselmo et al. to delay the exponential growth phase of B. coagulans by 15 h.3 In this article, we concentrate on the encapsulation of S. epidermidis in shells of increasing thickness made by LbL assembly, using either a pair of synthetic polyelectrolytes or a pair of oppositely-charged polysaccharides. We study the impact of shell composition, its thickness, the charge of the last deposited layer, and the aggregation state of the cells on viability and on the delay of growth. Aggregation state is varied through the use of different coating procedures – among which is a new scalable process that generates non-aggregated bacteria. We show for the first time that both the p. 5 ACS Paragon Plus Environment


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aggregation state and charge density of the external layer play a key role in the delay of bacterial growth, in addition to more expected parameters such as the shell thickness and the synthetic or polysaccharidic nature of the polyelectrolytes. We explain these findings based on our current understanding of the dynamic character of LbL assemblies and of the bacteriostatic effect of polyamines, and demonstrate that the mechanical properties of the LbL shell is not a dominant parameter. Using our new methodology to avoid the aggregation of coated bacteria, and the proper selection of LbL shell parameters, delays of the growth of up to 30 h can be obtained for a shell comprised of only three bilayers (thickness of ca. 7 nm), which is compatible with daily topical applications.

2. Experimental Section Materials and bacteria (Scheme 1): Poly(styrene sulfonate) (PSS, average molar mass by weight 70 000 g/mol) and poly(allylamine hydrochloride) (PAH, average molar mass 450 000) were purchased from Aldrich. They were dissolved in 0.15 M NaCl/0.005 M CaCl2/pH 7 (1 mg/mL). Chitosan (CHI, Protosan UP CL114 50 000-150 000 g/mol) was obtained from Novamatrix, and alginate (ALG, Ref-W201502, viscosity 5-40 cps at 1%, 25°C) was from Aldrich. They were dissolved in 0.15 M NaCl/pH 6 (1 mg/mL). Agarose (ultra-low gelling temperature, Aldrich A5030, melting point < 50°C, gel point 8-17°C at 0.8%) was dissolved in NaCl 0.15 M/pH 6 (10 mg/mL). All solutions were sterilized in an autoclave. NaCl (BioXtra, >99.5%) and CaCl2 (>93%) were from Aldrich. All aqueous solutions were prepared using deionized water with a resistivity of 18.2 MΩ.cm at 25°C. The alamarBlue® cell viability reagent and the LIVE/DEAD® BacLight™ Bacterial Viability Kit were purchased from Thermo-Fisher Scientific.


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Freeze-dried S. epidermidis was obtained from ATCC (ref. number 12228). The bacteria were revived by overnight incubation in vented culture tubes at 37°C/250 rpm in 5 mL broth (BD Difco™ Nutrient Broth 234000), transferred to an air-vented culture flask containing 700 mL of broth and cultured at 37°C/250 rpm until half exponential phase (optical density (OD) of ~0.55 measured at 540 nm in cells of 1 cm path length). 40 mL of bacterial culture were centrifuged and the supernatant was discarded. Then 10 mL of fresh broth was added in the tube and the bacterial pellets were resuspended by vortexing. 10 mL of a 50/50 v:v water/glycerol autoclaved mixture was added in the tube which was subsequently stored at -80°C until further use. Bacterial culture, purification and concentration (starting suspensions): The cryopreserved bacteria were revived overnight in 5 mL of broth in four 14 mL vented culture tubes (250 rpm/37°C). The pre-culture was placed in 700 mL broth in a vented culture flask, and grown at 37°C/250 rpm until an OD of ~0.55 (540 nm, cells of 1 cm path length). The bacteria were washed in 0.15 M NaCl at pH 6, concentrated by a factor of 20 by three successive steps of centrifugation/redispersion in NaCl 0.15 M at pH 6, and stored in the fridge at 4°C. This concentrated bacteria suspension will be henceforth called starting suspension. For the agarose-based coating process, 1 mL of the starting bacteria suspension was centrifuged, the supernatant was discarded, and the bacteria were dispersed in 2 mL of 1% agarose solution; the solution was gelled overnight in the fridge in 14 mL closed tubes. Coating the bacteria by the standard LbL process (Scheme 2a): 8 mL of the starting bacteria suspension was centrifugated and the supernatant was discarded; then, 2 mL of 0.15 M NaCl aqueous solution (at pH 6 for (CHI/ALG) coatings or pH 7 for (PAH/PSS) coatings) was added and the bacteria were suspended again by shaking/vortexing. 3 mL of polyelectrolyte solution was then added, and the bacteria were incubated for 10 min. p. 7 ACS Paragon Plus Environment


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Next, the bacterial suspension in polyelectrolyte was centrifuged at 4000 rpm for 210 min, the supernatant was removed, and the bacteria were dispersed again in 8 mL 0.15 M NaCl; this rinsing procedure was repeated thrice. The whole process was repeated until the desired number of layers, the first adsorbed layer being either CHI or PAH. The coated bacteria were finally diluted in 0.15 M NaCl by a factor of ten, and stored in the fridge at 4°C. Coating of the bacteria by the agarose process (Scheme 2b): 2 mL of polyelectrolyte (cold solution at 4°C) was added to 2 mL of the bacteria in agarose gel, and the tube was shaken moderately to break the gel and form dispersed particles of sub-mm size. After incubation for 10 min in melting ice, the tube was centrifuged at 0°C to form the gel again, and the supernatant was discarded. The collected gel was rinsed thrice following a similar procedure (qsp 10 mL of 0.15 M NaCl). The whole process was reiterated until the desired number of adsorbed layers. The sample was collected and stored in the fridge until further use. If need be, the gel can be dissolved by transferring 2 mL of bacteria in gel to 37 mL of heated 0.15 M NaCl (37°C); after 30 min at 37°C, the suspension is centrifuged, the supernatant discarded, and the bacteria resuspended in 0.15 M NaCl at room temperature. Determination of bacterial viability: 0.3 µL of Live/Dead® BacLightTM mixture kit (SYTO® 9 (3.34 mM)/propidium iodide (20 mM) 50:50 v:v) was added to 0.1 mL of bacterial suspension in 0.15 M NaCl. After a gentle shaking, the suspension was incubated for 20 min in the dark. The stained bacteria were then observed with a Zeiss Axiovert 200 M epifluorescence microscope at 40x magnification. The red and green channel images were thresholded in Wavemetrics Igor Pro; the intersection between red and green channels was obtained by multiplication after thresholding. The ratio between the number of purely green pixels and the total number of red or green pixels p. 8 ACS Paragon Plus Environment

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was defined as the percentage of living bacteria. The operation was repeated for a range of thresholds, and the average value obtained was taken as the final value. Determination of the lag times for growth: 0.889 mL of coated bacterial suspension was added to 8 mL of broth in 14 mL vented culture tubes, and the system was incubated in duplicate for varying amounts of time at 37°C and 250 rpm. For each time point, 0.25 mL of culture was placed in a well of a 96 well plate and the OD was measured at 540 nm with a Tecan plate reader. Then, 25 µL of alamarBlue® was added in the well and the fluorescence at 585 nm was measured under excitation at 570 nm (bandwidth of 5 nm). After incubation for 30 min at 37°C, the fluorescence was measured again. The difference of fluorescence between the two measurements defines the metabolic activity. The collected OD data from the two experiments were combined together when superimposing, otherwise they were treated separately. The data were interpolated with a smoothing-spline routine, and the highest slope in the exponential growth phase was obtained by differentiation. The intercept of the line of highest slope with the zeroculture time base line was defined as the onset of growth.

ζ-potential measurements: A Folded Capillary cell DTS1070 from Malvern Instruments was first flushed with 0.15 M NaCl (pH 6) then filled with a diluted bacterial suspension. The zeta measurements were performed at 25°C with a Zetasizer Nano ZS (Malvern Instruments).

3. Results 3.1. Coating the bacteria The bacteria were coated by LbL adsorption of pairs of polyelectrolytes of opposite charge (Scheme 1), either poly(allylamine hydrochloride)/poly(styrene sodium p. 9 ACS Paragon Plus Environment


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sulfonate) at pH 7 or chitosan/alginate at pH 6, both in 0.15 M NaCl, leading to shells of formula (PAH/PSS)n and (CHI/ALG)n respectively, where n is the number of bilayers.

Scheme 1. Structure of the polyelectrolytes used to encapsulate the bacteria.

The polyelectrolyte multilayer growth in our conditions is based on electrostatic interactions, and was determined to be linear by performing stylus profilometry on flat films deposited on glass slides, with a dry thickness increment per bilayer of 2.3 nm for PAH/PSS and of 2.2 nm for CHI/ALG (Supporting Information Figure S1). Hence, the amount of matter adsorbed per adsorption cycle is essentially identical for the two systems, allowing us to perform meaningful comparisons between a synthetic and a polysaccharide-based system. It should be realized, however, that the two systems swell very differently when immersed in water, due to the different strength of interaction between their charged groups60 and the different hydrophilicity of their chain backbones: (PAH/PSS)-based multilayers were reported to swell by ca. 30% in water,61 while the swelling is larger than 100% for (CHI/ALG) multilayers.62 As a consequence, their Young's moduli in aqueous conditions differ by one order of magnitude, ca. 600 MPa for (PAH/PSS)n,61 and ca. 30 MPa for (CHI/ALG)n.62


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Scheme 2. Scheme of the bacteria coating processes, (a) either by the standard LbL route which favors bacterial aggregation, or (b) by the agarose gel route in which aggregation is avoided and supernatant removal is made easier (see also Supporting Information Figure S3 for pictures of the agarose route).

Different processes were used to encapsulate the bacteria. In a first, standard process (Scheme 2a), the bacteria were immersed in solutions of the polycation and polyanion in turn, with three intermediate rinsing steps in a 0.15 M NaCl aqueous solution between the adsorption of each layer. Purification of the bacteria by centrifugation was performed after each adsorption or rinsing step. ζ-potential measurements performed after the adsorption of the first layers (Figure 1, first two top panels) indicate that the bacterial charge oscillates as expected between positive and p. 11 ACS Paragon Plus Environment


negative values after the adsorption of the polycation and of the polyanion, respectively. However, the first adsorption step of chitosan was found not to be able to overcompensate the starting negative charge of the bare bacteria membrane. The difference of charge overcompensation seen for the synthetic versus polysaccharide system is a direct consequence of the lower linear charge density of the polysaccharides (ca. two charges per nm, as opposed to four per nm for the synthetic polyelectrolytes).

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CHI/ALG, standard process

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Figure 1. Zeta potential of the bacteria after the adsorption of the polyelectrolyte shell (n bilayers), for the standard and agarose-based processes. Non-integral values of the number of bilayers correspond to a polycation-ended shell.

As a consequence of the incomplete charge overcompensation after the first adsorption of chitosan, strong aggregation was observed for the bacteria coated by the polysaccharides in the standard process (Figure 2). Microscopy observations performed at each step of the process indicated that aggregation occurred during the centrifugation step immediately following the first adsorption of chitosan (Supporting Information Figure 2). Aggregation was also observed for PAH/PSS-coated bacteria, but to a much p. 12 ACS Paragon Plus Environment

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smaller extent: aggregates typically comprised a few tens of bacteria for PAH/PSS shells, while aggregates of many hundreds of bacteria were observed for the CHI/ALG shells (Figure 2). Although not always acknowledged, aggregation is a common problem for the LbL coating of living cells.52 This issue is probably even more critical for S. epidermidis, bacteria which were demonstrated to readily aggregate in salted water.63 It most likely arises from the presence of both positive and negative charges on the outer membrane of the cell, which favors short range attraction, resulting in a negative excluded

volume.

This

is

certainly

the

case

for

the

incompletely-charge

overcompensated bacteria after the adsorption of the first chitosan layer.

Figure 2. Overlay (red/green) epifluorescence optical micrographs of S. epidermidis coated by multilayers of increasing number of bilayers (n), using the standard LbL process involving purification by centrifugation after adsorption. Considerable aggregation occurs when growing (CHI/ALG) multilayers, contrarily to (PAH/PSS) multilayers. The bacteria were stained with the Live/Dead® BacLightTM kit and appear green when alive, red when dead.

Different methods have been proposed to avoid aggregation when coating colloids. One possible solution is to vary colloid and polyelectrolyte concentrations with aims of p. 13 ACS Paragon Plus Environment


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minimizing the probability of bridging and of chance collisions, as previously demonstrated for the coating of red blood cells.52 However, no such conditions were found for S. epidermidis. Another possibility is to avoid the centrifugation step by removing the excess polyelectrolyte by filtration and/or osmosis.64 We adapted this process to S. epidermidis, by dialyzing the suspension of bacteria in chitosan against 0.15 M NaCl, using a membrane of cutoff above the size of the chitosan coils (Supporting Information Figure 2b); the bacteria were kept in suspension above the membrane under constant agitation to prevent sedimentation (which was found to also lead to aggregation). Although successful (Supporting Information Figure 2a), the process was slow, typically requiring 12 h of dialysis to remove the excess chitosan after the first adsorption step. Additionally, the concentration of bacteria had to be decreased to avoid aggregation by random collision, resulting in low throughput. Therefore, we modified a method initially proposed by Richardson et al.,65 in which colloids to be coated are embedded in a block of agarose gel. Because agarose forms a nano-porous gel,66 diffusion of polyelectrolyte chains through the gel is possible, whereas the colloids are effectively trapped within the gel. Here, we used an ultra-low gelling temperature agarose, which remains soluble at room temperature when cooled from the soluble phase, allowing for the addition of bacteria without thermal shock. The bacteria/agarose mixture can then be gelled at 4°C. We then used the self-healing properties of this physical gel to speed-up the coating/rinsing process, which was entirely conducted at 4°C (Scheme 2b and Supporting Information Figure S3). After adding the adsorption or rinsing solution, the gel is gently shaken and fragments in small particles of sub-mm size in which the bacteria are trapped. Diffusion of the polymer chains is rapid in this dispersed geometry (adsorption and rinsing times of 10 and 3 min were used, respectively). The suspension p. 14 ACS Paragon Plus Environment

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of microgels is then centrifuged, leading to the formation of a new block of gel in the tube due to H-bonding interactions between the sub-mm particles of gel. Elimination of the supernatant is then trivial, and the process can be easily repeated. The gel can be dissolved at 37°C if diluted by a factor of ten or more, due to the variation of the gelling temperature with concentration. For culture experiments, this step was not necessary, as dissolution simultaneously occurred in the culture medium at 37°C. With this process, it became possible to essentially eliminate bacterial aggregation, as shown in Figure 3, while coating a large amount of bacteria in a reasonably limited time.

ζ-potential

measurements

(Figure

1,

bottom

panel)

indicated

proper

overcompensation of the charge of the coating after each adsorption step. Interestingly, the adsorption of a single layer of chitosan is now sufficient to overcompensate the negative charge of the bacteria, whereas this was not the case in the absence of the agarose gel. This suggests that the chitosan chains adsorb in more loopy conformations in the agarose process, with therefore a more positive global charge of the layer at saturation. This might be due to the confinement of the chains in the pores of the agarose gel; confinement of chains in pores has been demonstrated before to change layer thicknesses in LbL.67,68



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Figure 3. Overlay (red/green) epifluorescence optical micrographs of S. epidermidis coated by (CHI/ALG) multilayers of increasing number of bilayers (n), using the agarose process involving adsorption through agarose gel sub-mm particles entrapping the bacteria. Bacterial aggregation is fully suppressed, and the process is made easier compared to standard LbL. The bacteria were stained with the Live/Dead® BacLightTM kit and appear green when alive, red when dead; the lateral size of each image is 100 µm.

To have a more quantitative evaluation of the interest of the agarose process, the distribution of the area of the bacterial aggregates when seen by epifluorescence microscopy was computed for a series of samples. The distributions by area are plotted in Figure 4, showing that the agarose process preserves the distributions of area within experimental accuracy, confirming the successful elimination of aggregation. In contrast, the standard process results in a shift of the area distributions by a factor of ca. 10 and 100 for (PAH/PSS) and (CHI/ALG) shells, respectively.


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+(PAH/PSS)6 agarose process +(CHI/ALG)6 agarose process Starting bacteria -2

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Figure 4. Distribution (by area) of the area covered by bacteria and bacterial aggregates, for starting bacteria and bacteria covered by LbL shells of large number of bilayers, fabricated by the agarose and standard processes. The distributions are shifted vertically for clarity.

With this new procedure, the processing time per bilayer was typically from 1h to 1h30 depending on the details of the setup, which is standard for the LbL coating of hard colloids. Additionally, we generally processed seven 15 mL tubes in parallel, each containing 2 mL of bacteria-loaded gel made from 20 mL of starting culture. This corresponds to the parallel processing of 140 mL of bacterial culture. If we extrapolate these numbers to larger 50 mL tubes, the processing of up to 500 mL starting bacterial culture would be feasible with only 3-4 such tubes and standard laboratory equipment, which is a significant throughput. The viability of the bacteria was evaluated for a series of coating experiments, using a Live/Dead® BacLightTM bacterial viability kit. Only samples with limited aggregation could be analyzed in this way. As no effect of the number of deposition cycles on viability p. 17 ACS Paragon Plus Environment


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could be observed except between non-coated and coated bacteria (Supporting Information Figure S4), the data obtained for bacteria coated by shells of different thickness were aggregated (Figure 5). The collection and purification of the bacteria before coating results in an average of 77% live bacteria in the sample (with a standard error of 2.6%). When coated, the average viability decreases to 59% (with a standard error of 2%) for all samples included, except bacteria coated with PAH/PSS by the agarose process which appear to survive statistically significantly better (average viability of 75% with a standard error of 3%). These experiments thus show that the coating processes are only moderately influencing the viability of the cells, with no significant effect of the number of coating cycles. Overall, roughly 25% of the bacteria are killed upon collection, with a further 20% destroyed upon coating, all processes and shells included except (PAH/PSS) with the agarose process.

Figure 5. Average viability of the bacteria before and after the coating process. The error bar is the standard error; stars indicate significance levels of 0.05 (*) and 0.001


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(***). The data corresponding to different shell thicknesses (Supporting Information Figure S4) were aggregated.

3.2. Bacterial growth The coated bacteria were cultured in broth, starting from suspensions of approximately equal optical density. The growth was followed by measuring the OD as a function of time; in addition, the metabolic activity was evaluated by a colorimetric assay based on the reduction of resazurin into resorufin (alamarBlue®). All experiments were performed in duplicate, and merged together for analysis. In addition, a few tests were performed twice or thrice to check reproducibility, starting from different bacterial cultures. Typical growth curves are shown in Figure 6. After some time lag which increases with the coating thickness, the bacteria enter exponential growth, followed by saturation and death. The metabolic assay gives similar results as the OD data although sampled at less frequent time points; therefore, it was not used further in subsequent experiments. The lag time was defined on the OD curve as the time corresponding to the intercept of the baseline with the line of highest slope in the exponential growth regime (Figure 6). Prior to these measurements, we checked that the lag times are only moderately dependent on the starting OD, with a delay of only ca. 3 h per decade of dilution in the experimental range of our experiments (Supporting information Figure S5). This ensures that the slight variations of concentration due to losses during purification and coating are not responsible for significant shifts in the lag times, when comparing different samples. Finally, the corrected lag time (hereafter simply called 'lag time') was obtained as the difference between the lag times of the coated and uncoated bacteria measured in a same run. p. 19 ACS Paragon Plus Environment

OD (540 nm)

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Culture time (h)

Figure 6. Evolution of the optical density (OD) at 540 nm and of the metabolic activity (alamarBlue® assay) during the culturing of S. epidermidis coated by n CHI/ALG bilayers through the agarose gel procedure. The lag times are indicated by the thin vertical lines; they were determined as the intersections of the dashed lines drawn on the OD curves. The curves are shifted vertically for clarity, by 1.2 and 12000 (n=0), 0.8 and 8000 (n=1), and 0.4 and 4000 (n=2), for the OD and metabolic activity, respectively.

The lag times are collected in Figure 7 for polyanion-ended shells (PSS or ALG), and in Figure 8 for polycation-ended shells (PAH or CHI). Open symbols in red indicate samples for which no growth occurred over at least 60 h. Live/Dead staining performed after rinsing out the broth on such non-growing coated bacteria indicated all bacteria to be dead (Supporting Information Figure S6), although they were living before incubation in the broth. p. 20 ACS Paragon Plus Environment

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Lag time (h)

agarose process

standard CHI/ALG process

40 20 0

PAH/PSS No growth

Lag time (h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 20 0 0

2 4 6 80 2 4 6 8 Number of bilayers Number of bilayers

Figure 7. Lag times for the growth of S. epidermidis coated with polyanion-ended shells made of (CHI/ALG)n (top) or (PAH/PSS)n (bottom), using either the standard LbL process (right) or coating through an agarose gel (left). Open symbols in red indicate that no growth occurred over at least 60 hours. Vertical bars connect points obtained in different experiments.



Lag time (h)

agarose process

standard CHI/ALG process No growth

60 40 20 0

PAH/PSS Lag time (h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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No growth

60 40 20 0 0

2

4

6

Number of bilayers

80

2

4

6

8

Number of bilayers

Figure 8. Lag times for the growth of S. epidermidis coated with polycation-ended shells made of (CHI/ALG)n (top) or (PAH/PSS)n (bottom), using either the standard LbL process (right) or coating through an agarose gel (left). Open symbols in red indicate that no growth occurred over at least 60 hours. Vertical bars connect points obtained in different experiments.

The lag times differ substantially depending on the number of bilayers, on the coating process and therefore the state of aggregation of the bacteria, on the charge of the outermost layer, and on the composition of the shell. For polyanion-ended shells (Figure 7), an increasing number of bilayers in the shell initially leads to a quasi-linear increase of the lag time. For more than 2-3 bilayers, the lag time of non-aggregated (CHI/ALG)-coated bacteria (prepared by the agarose process) saturates to a value on the order of 30 h, whereas strongly aggregated (CHI/ALG)-coated bacteria (prepared by the standard process) display a significantly lower lag time at saturation (15 h). (PAH/PSS)coated bacteria behave differently, with a large dispersion of lag times for more than 2-3 p. 22 ACS Paragon Plus Environment

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bilayers, and frequently no growth being observed due to bacterial death in the culture medium at 37°C as noted before. Polycation-ended shells (Figure 8) always have a stronger impact on the growth of bacteria, with larger and more dispersed lag times than polyanion-ended ones, and bacterial death very frequently observed at 37°C in the broth, with the exception of aggregated (CHI/ALG)-coated bacteria. Figure 9 shows that, for (CHI/ALG) shells, the lag time increases each time a positively-charged layer is added as the final layer on the shell; in contrast, it decreases when a supplementary negatively-charged layer is added to the shell. For bacteria coated by the agarose process, the effect is even more visible and can still be seen for five bilayers, i.e., when the last layer is more than 10 nm away from the bacteria walls. A similar trend is observed for (PAH/PSS) shells, although it is less prominent due to the lower viability of (PAH/PSS)-coated bacteria in the broth at 37°C.

CHI/ALG

PAH/PSS No growth

60 standard process

40

Lag time (h)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


standard process

20 0

No growth

60 40 20

agarose process

agarose process

0 0

2

4

6

Number of bilayers

8 0

2

4

6

8

Number of bilayers

Figure 9. Lag times for the growth of S. epidermidis coated with shells made of (CHI/ALG)n (left) or (PAH/PSS)n (right), using either the standard LbL process (top) or



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coating through an agarose gel (bottom). Open symbols in red indicate that no growth occurred over at least 60 hours. Vertical bars connect points obtained in different experiments. A semi-integral number of bilayers corresponds to a polycationic external layer (open symbols). Dashed black lines are drawn to highlight the "odd/even" effect of the shells.

4. Discussion Differences in lag times might a priori originate from purely physical reasons, such as the mechanical rigidity of the shell, or the delayed diffusion of nutrients through the shell. However, a mechanical effect can be safely ruled out, since the lag times of (CHI/ALG) and (PAH/PSS) shells are in the same range (Figure 7) despite their Young's moduli differ by a factor of ten when swollen (literature values61,62), whereas the amount of polymer adsorbed per bilayer is similar. In support of this conclusion, we also performed an experiment in which the bacteria were coated by the agarose process, first by 2.5 soft layers of (CHI/ALG), then by up to 2.5 layers of stiffer (PSS/PAH); the lag times were not increased compared to bacteria coated by soft (CHI/ALG) layers only. As for the increased diffusion time of nutrients through the shell, it would result in a time lag proportional to the square of the shell thickness (or of the number of bilayers), which is not observed. In addition, the significantly higher swelling of (CHI/ALG) shells in water should result in shorter diffusion times and therefore shorter time lags of growth, which is again not observed. Hence, it is unlikely that hindrances to nutrient diffusion be the dominant factor governing the lag times. However, the "odd/even" effect shown in Figure 9 provides an important clue as to the origin of the increased lag times, because it directly relates to the evolution of the shell composition. Due to charge overcompensation, polycation-ended coatings have a p. 24 ACS Paragon Plus Environment

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surface excess of ammonium groups, which are not paired with carboxylate groups of the polyanions. These unpaired ammonium groups (or extrinsic sites in the terminology of Schlenoff and coworkers69) can diffuse throughout the film by dynamic exchange with paired sites, as recently shown.69 This diffusion process does not involve macromolecular motion and just requires short-range segmental motion – in a kind of 'flipping-around' mechanism.69 When the coated bacteria are immersed in the broth at 37°C, this mechanism is accelerated and the layer equilibrates. As a result, the amount of unpaired ammonium groups also increases at the bacterium surface during culture at 37°C. Polyamines are generally cytotoxic,70 which is due to their de-structuring interaction with

the

cell

membrane,

and/or

to

interference

with

specific

macromolecules involved in the bacterial growth process; therefore, the bacterial growth of polycation-ended layers is strongly delayed or even entirely arrested. Adsorption of a supplementary polyanionic layer on the shell reverses the situation, with the addition of an excess of innocuous negatively-charged carboxylic acid groups. In the broth at 37°C, these groups pair with extrinsic ammonium sites throughout the whole film via the extrinsic site diffusion (exchange) mechanism; the resulting paired groups are less detrimental to growth, leading to a limited bacteriostatic effect. Therefore, the time lags arise from a bacteriostatic or bactericidal effect resulting from the composition of the shell in paired and unpaired amine/ammonium groups, which depends on the nature of the last adsorbed layer. Additionally, this hypothesis also explains why the lag times increase over the 2-3 first adsorption cycles, then saturate: this is because, as proposed by the zone-model of polyelectrolyte multilayers,71 the average composition of the bottom part of the coating evolves over the first cycles of adsorption of the LbL process, then saturates to a constant value. Furthermore, due to a larger amount of amine groups, PAH-based multilayers are more bactericidal than CHIp. 25 ACS Paragon Plus Environment


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based multilayers, and frequently fully destroy the bacteria in the broth at 37°C as seen in Figure 8. Finally, when the bacteria strongly aggregate as is the case for (CHI/ALG) shells made by the standard LbL procedure, the aggregates shield their inner bacteria from the adverse effect of the excess polyamine, resulting in a smaller bactericidal activity (right top panel of Figure 8). Before concluding, it is interesting to note that the lag times observed here at saturation are similar to the germination delays observed for yeast coated by comparable systems and to the 15 h growth delay reported for B. coagulans coated by three bilayers of (glycol chitosan/ALG).3 This suggests that similar mechanisms could be playing a role for these cases as well.

5. Conclusions LbL assembly can be used to control the growth of bacteria, based on the bacteriostatic effect of a properly ion-paired polyelectrolyte complex. The mechanical properties of the coating do not play a significant role, nor do the hindrances to nutrient diffusion, at least in the range of thicknesses probed in this study. It is thus not needed to add a large number of adsorbed bilayers to delay growth, since a few layers are sufficient to generate this effect. However, it is critical to control the polyelectrolyte selected for the outmost layer, to avoid an excess of unpaired amine groups, which leads to bacterial death. Other parameters can be used to control the composition of the coating and its degree of ion-pairing, as is well-known in the LbL field; these could be harnessed to further fine-tune the bacteriostatic activity of LbL shells. Two other parameters are critical for the control of the bacteriostatic effect. The first is the state of aggregation of the bacteria, which needs to be avoided for maximal effects. Here, we developed a new coating method which fully suppressed aggregation p. 26 ACS Paragon Plus Environment

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and eased the coating process, and operates at the scale of 150 mL starting bacterial culture. Importantly, this methodology could easily be scaled up to process with standard laboratory equipment larger amounts of bacteria, in the range of 500 mL of starting bacterial culture. The second critical parameter is the composition of the shell, with polysaccharide-based coatings being superior to coatings based on synthetic polyelectrolytes which tend to be more bactericidal. The lag times reported here would typically be sufficient for preventing proliferation in daily topical applications. Considering the low number of layers needed, the significantly-improved methodology of coating based on agarose gels, and the rich catalog of possible polysaccharides, the LbL encapsulation of commensal skin bacteria appears to be a viable solution.



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Associated content The Supporting Information is available free of charge on the ACS Publications website 1. Thickness of the multilayers 2. Identification of the step responsible for the aggregation of the bacteria, and elimination of the aggregation by dialysis instead of centrifugation 3. Pictures of the agarose gel procedure 4. Viability of the coated bacteria versus shell thickness 5. Effect of dilution on the time lag for growth 6. Evaluation of the viability of cultured non-growing bacteria



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Author information Corresponding Author *E-mails: [email protected]; [email protected]. Phone: +32 10 47 37 65. Notes The authors declare no competing financial interest

Acknowledgements The authors thank Lisa Freed and Zhihao Li for helpful discussions. Financial support was provided by the Fulbright Scholar Program (A.M.J.), the Louvain Fundation (A.M.J.), Wallonia-Brussels International (K.G.), and the FNRS (A.M.J. and K.G.). K.G. is Research Associate of the F. R. S.–FNRS.


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Graphic for manuscript (TOC)


Controlling the Growth of Staphylococcus epidermidis by Layer-By

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